Method for measuring pad and method for diagnosing disease using same

Abstract

The present disclosure addresses the problem of providing a method for measuring PAD activity.　Provided are: a method for measuring PAD activity in a sample, the method comprising a step in which the sample is brought into contact with a substrate peptide to generate a peptide containing an L-citrulline residue, the generated peptide containing an L-citrulline residue is subsequently digested using a peptidase to release L-citrulline, and the released L-citrulline is then oxidized with citrulline oxidase and measured; and a composition, a reagent, and a kit for the method. Also provided are a composition, a reagent, and a kit for measuring PAD activity that each contain a substrate peptide. Also provided are a modified citrulline oxidase and a composition, a reagent, and a kit for measuring PAD activity that each contain the modified citrulline oxidase. Also provided are a composition, a reagent, and a kit for diagnosing rheumatism. Also provided are a composition, a reagent, and a kit for diagnosing Alzheimer's disease.

Description

PAD measurement method and disease diagnostic method using the same

[0001] The present invention relates to a PAD measurement method and a method for diagnosing diseases using the same, such as a method for diagnosing rheumatoid arthritis, Alzheimer's disease, cancer, ulcerative colitis, inflammatory bowel disease, fibrosis, and multiple sclerosis.

[0002] Although L-citrulline is an amino acid, it is not included in the so-called 20 natural amino acids specified by triple codons. L-citrulline production pathways include the conversion of L-arginine residues in polypeptides to L-citrulline by peptidylarginine deiminase (PAD), and the conversion from L-ornithine by ornithine transcarbamoylase in the ornithine cycle. Abnormal L-citrullination of biological proteins has been reported to be associated with various diseases, such as rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, prion diseases, hepatic fibrosis, psoriasis, chronic obstructive pulmonary disease, and cancer.

[0003] Rheumatoid arthritis is a representative inflammatory autoimmune disease. The conventional diagnostic method for rheumatoid arthritis is based on rheumatoid factor (RF). However, RF can produce false positives even in healthy individuals. Another diagnostic method is colorimetric analysis.

[0004] Another diagnostic method for rheumatoid arthritis involves the use of anti-cyclic citrullinated peptide antibodies (anti-CCP antibodies). Anti-CCP antibodies show positive results earlier than RF in patients with rheumatoid arthritis and have higher specificity, making them useful in diagnosing rheumatoid arthritis. However, in early stages of rheumatoid arthritis, approximately 30% of tests using anti-CCP antibodies can produce false negatives. Furthermore, because it uses an immunological assay method, it has drawbacks such as long measurement times and high testing costs.

[0005] Therefore, instead of measuring anti-CCP antibodies, a method has been proposed to measure the citrullinated peptide itself. For example, Patent Document 1 describes a method in which L-citrulline is converted to pyrophosphate by the action of argininosuccinate synthase, then pyrophosphate pyruvate dikinase is applied to the pyrophosphate to convert it to pyruvate, and the amount of pyruvate obtained is calculated to quantify L-citrulline.

[0006] Furthermore, Patent Document 2 describes a method for quantifying L-citrulline, an oxidoreductase for quantification, a composition for quantification, a kit for quantification, and a method for evaluating the activity of peptidylarginine deiminase. The disclosed oxidoreductase is prepared based on arginine oxidase derived from Pseudomonas.

[0007] On the other hand, there are five types of peptidylarginine deiminase (PAD) known, broadly classified as PAD1, PAD2, PAD3, PAD4, and PAD6. Of these, PAD2 has been suggested to be associated with Alzheimer's disease, and PAD4 with the onset of rheumatoid arthritis (Non-Patent Literature 1). In addition, PAD4 has been reported to be associated with fibrosis and cancer (Non-Patent Literature 3). Furthermore, PAD4 has been reported to be associated with multiple sclerosis (Non-Patent Literature 4). Furthermore, PAD4 has been reported to be associated with cancer (Non-Patent Literature 5). Furthermore, PAD4 has been reported to be associated with fibrosis (Non-Patent Literature 6). Furthermore, PAD4 has been reported to be associated with ulcerative colitis (Non-Patent Literature 7). Furthermore, PAD4 has been reported to be associated with Alzheimer's disease (Non-Patent Literature 8). Furthermore, PAD2 has been reported to be associated with inflammatory bowel disease (Non-Patent Literature 3). Furthermore, PAD2 has been reported to be associated with multiple sclerosis (Non-Patent Literature 9). In addition, PAD2 has been reported to be associated with cancer (Non-Patent Literature 5). To diagnose these diseases, in addition to approaches that quantify citrullinated peptides contained in a sample, it is also desirable to measure the PAD activity itself, which converts peptides containing L-arginine residues into peptides containing L-citrulline residues.

[0008] Patent document 3 describes L-glutamate oxidase. Non-patent document 2 describes the substrate specificity of PAD2 and PAD4.

[0009] Japanese Patent Publication No. 2013-162752 (Japanese Patent No. 5303715), International Publication No. 2021 / 054375, International Publication No. 2024 / 090562

[0010] Akihito Ishigami, "Citrulline Molecules and Geriatric Diseases," Journal of the Japan Geriatrics Society, Japan Geriatrics Society, July 2014, Vol. 51, No. 4, p. doi.org / 10.1038 / s41598-025-24221-2.Journal of Neuroscience 1 November 2006, 26 (44) 11387-11396Mol Cancer Res. 2025 Oct 2;23(10):844-858. doi: 10.1158 / 1541-7786.MCR-24-1095.Circ Res. 2019 Aug 15;125(5):520-522. doi: 10.1161 / CIRCRESAHA.119.315601Clin Immunol. 2008 Feb;126(2):165-71. doi: 10.1016 / j.clim.2007.09.001. Epub 2007 Nov 5.May 2012 Journal of Autoimmunity 38(4):369-80. DOI:10.1016 / j.jaut.2012.03.004Dis Model Mech. 2008 Nov-Dec;1(4-5):229-40. doi: 10.1242 / dmm.000729. Epub 2008 Nov 6.

[0011] In some embodiments, the object of this disclosure is to provide a method for measuring PAD activity, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing rheumatoid arthritis, or a method for assisting in the diagnosis of rheumatoid arthritis, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing Alzheimer's disease, or a method for assisting in the diagnosis of Alzheimer's disease, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing multiple sclerosis, or a method for assisting in the diagnosis of multiple sclerosis, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing cancer, or a method for assisting in the diagnosis of cancer, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing fibrosis, or a method for assisting in the diagnosis of fibrosis, or a composition, reagent, or kit for that purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing ulcerative colitis, or a method to assist in the diagnosis of ulcerative colitis, or a composition, reagent, or kit for the same purpose. In some embodiments, the object of this disclosure is to provide a method for diagnosing inflammatory bowel disease, or a method to assist in the diagnosis of inflammatory bowel disease, or a composition, reagent, or kit for the same purpose. In some embodiments, the object of this disclosure is to provide a substrate peptide for measuring PAD activity, or a composition, reagent, or kit for measuring PAD activity containing the substrate peptide. In some embodiments, the object of this disclosure is to provide citrulline oxidase for measuring PAD activity, or a composition, reagent, or kit for measuring PAD activity containing citrulline oxidase. In some embodiments, the object of this disclosure is to provide a method for quantifying citrulline.

[0012] As a result of diligent research to solve the aforementioned problems, the present inventors have found, as an example, that a peptide having a specific amino acid residue can serve as a substrate peptide for peptidylarginine deiminase (PAD), and have completed the present invention which includes this as one embodiment (as an example, a substrate peptide for PAD4). Furthermore, as an example, they have found that a peptide having a specific amino acid residue can serve as a substrate peptide for PAD2, and have completed the present invention which includes this as one embodiment.

[0013] Furthermore, as a result of diligent research to solve the aforementioned problems, the present inventors have found that, as an example, an enzyme having citrulline oxidase activity can be produced by substituting a specific amino acid residue in glutamate oxidase derived from the genus Streptomyces with another specific amino acid residue, and have completed the present invention which incorporates this as one embodiment.

[0014] Furthermore, as a result of diligent research to solve the aforementioned problems, the present inventors have found that, as an example, in the first step, the substrate peptide is brought into contact with a sample, and the PAD activity contained in the sample converts the L-arginine residue in the substrate peptide to an L-citrulline residue; in the second step, the peptide having the L-citrulline-converted residue is treated with a peptidase to release L-citrulline; and in the third step, the PAD activity in the sample can be measured by measuring the released L-citrulline. The present inventors have completed the present invention, which includes a method comprising these three steps as one embodiment. Furthermore, as a result of diligent research to solve the aforementioned problems, the present inventors have found that, as an example, citrulline can be quantified by using the citrulline oxidase of this disclosure, and the present inventors have completed the present invention, which includes this as one embodiment.

[0015] The present invention encompasses the following embodiments: [1] A method for measuring peptidylarginine deiminase (PAD) activity in a sample, comprising the steps of: (i) contacting a sample with a substrate peptide containing an L-arginine residue to cause peptidylarginine deiminase (PAD) contained in the sample to act on the substrate peptide containing the L-arginine residue, thereby generating a peptide containing an L-citrulline residue; (ii) digesting the peptide containing the L-citrulline residue generated in step (i) using a peptidase to release L-citrulline; and (iii) oxidizing the L-citrulline released in step (ii) using a citrulline oxidase and measuring the activity. [2] The method according to Embodiment 1, wherein the PAD is PAD4. [3] The method according to Embodiment 2 for diagnosing rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, or for assisting in the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, wherein the sample is a biological sample obtained from a subject, the PAD4 activity of the biological sample is measured by the method according to Embodiment 2, and if the PAD4 activity is higher than a threshold, the method includes or assists in diagnosing the subject as having rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease. [4] The method according to Embodiment 1, 2, or 3, wherein the substrate peptide containing the L-arginine residue has an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].[5] The method according to Embodiment 4, wherein the substrate peptide containing the L-arginine residue consists of the amino acid sequence Met-Asp-Arg-Asp-Gly. [6] The measurement method according to Embodiment 1, wherein PAD is PAD2. [7] The method according to Embodiment 6 for diagnosing Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, or for assisting in the diagnosis of Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, wherein the sample is a biological sample obtained from a subject, the PAD2 activity of the biological sample is measured by the method according to Embodiment 6, and if the PAD2 activity is higher than a threshold, the method includes or assists in diagnosing that the subject has Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease. [8] The substrate peptide containing the L-arginine residue has an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; and the third amino acid residue is L-arginine (Arg). The method according to Embodiment 1, 6, or 7, wherein the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, and the fifth amino acid residue X5 is glycine. [9] The method according to Embodiment 1, 6, or 7, wherein the substrate peptide containing the L-arginine residue consists of the amino acid sequence Lys-Gln-Arg-Lys-Gly.

[10] The method according to any one of Embodiments 1 to 9, wherein the citrulline oxidase is a modified citrulline oxidase, the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, in the modified citrulline oxidase, one or more amino acids are substituted at positions corresponding to positions 291, 302, 554, or 221, or 502, 479, 305, 310, or 113 of SEQ ID NO: 1, and the modified citrulline oxidase has decreased reactivity to L-arginine and increased activity to L-citrulline compared to the original polypeptide.

[11] In the modified citrulline oxidase, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is substituted with glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase; the amino acid at the position corresponding to position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid in the modified citrulline oxidase; the amino acid at the position corresponding to position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at the position corresponding to position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. The method according to Embodiment 10, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine, and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

[12] A modified citrulline oxidase, wherein the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, and in the modified citrulline oxidase, one or more amino acids are substituted at positions corresponding to positions 291, 302, 554, or 221, or 502, 479, 305, 310, or 113 of SEQ ID NO: 1, and the modified citrulline oxidase has decreased reactivity to L-arginine and increased activity to L-citrulline compared to the original polypeptide.

[13] The amino acid at position 291 of SEQ ID NO: 1 is substituted with glutamine in the modified citrulline oxidase; the amino acid at position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase; the amino acid at position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine in the modified citrulline oxidase; the amino acid at position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid in the modified citrulline oxidase; the amino acid at position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. A modified citrulline oxidase according to Embodiment 12, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine, and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

[14] A modified citrulline oxidase according to any one of Embodiments 12 to 13, wherein the polypeptide before modification is a polypeptide derived from the genus Streptomyces.

[15] A modified citrulline oxidase according to any one of Embodiments 12 to 14, wherein FAD is present as a coenzyme.

[16] A composition for measuring PAD activity, comprising the modified citrulline oxidase according to any one of Embodiments 12 to 15.

[17] The composition according to Embodiment 16, comprising a substrate peptide containing an L-arginine residue having PAD4 and further an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].

[18] The composition according to Embodiment 17, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Met-Asp-Arg-Asp-Gly.

[19] PAD is PAD2, and furthermore, an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; and the third amino acid residue is L-arginine (Arg),

[20] The composition according to Embodiment 16, comprising a substrate peptide containing an L-arginine residue, wherein the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, and the fifth amino acid residue X5 is glycine.

[20] The composition according to Embodiment 19, wherein the substrate peptide containing the L-arginine residue consists of the amino acid sequence Lys-Gln-Arg-Lys-Gly.

[21] The composition according to any one of Embodiments 16 to 20, further comprising a peptidase.

[22] The composition according to Embodiment 21, comprising a plurality of peptidases.

[23] The composition according to Embodiment 22, wherein the plurality of peptidases are selected from the group consisting of (i) a first endopeptidase and a second endopeptidase, (ii) an endopeptidase and an exopeptidase, and (iii) a first exopeptidase and a second exopeptidase.

[24] A polynucleotide encoding the modified citrulline oxidase according to any one of Embodiments 12 to 15.

[25] A vector comprising the polynucleotide according to Embodiment 24.

[26] A host cell comprising the vector according to Embodiment 25.

[27] A method for producing modified citrulline oxidase, comprising the steps of (i) culturing the host cell according to Embodiment 25 to produce the modified citrulline oxidase according to any one of Embodiments 12 to 15, and (ii) obtaining the produced modified citrulline oxidase.

[28] A composition for measuring PAD4 activity by enzymatic method comprising a substrate peptide containing an L-arginine residue having an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].

[29] The composition according to Embodiment 28, comprising the amino acid sequence Met-Asp-Arg-Asp-Gly.

[30] A composition for the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, comprising the composition according to Embodiment 28 or 29, a peptidase, and citrulline oxidase.

[31] Amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, A composition for measuring PAD2 activity by enzymatic method, comprising a substrate peptide containing an L-arginine residue, having the fifth amino acid residue X5 being glycine.

[32] The composition according to Embodiment 31, wherein the substrate peptide containing the L-arginine residue comprises the amino acid sequence Lys-Gln-Arg-Lys-Gly.

[33] A composition for diagnosing Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, comprising the composition according to Embodiment 31 or 32, a peptidase, and citrulline oxidase.

[34] The composition according to any one of Embodiments 28 to 33, wherein the citrulline oxidase is a modified citrulline oxidase, the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is substituted with glutamine, and the modified citrulline oxidase has reduced reactivity to L-arginine and increased activity to L-citrulline compared to the original polypeptide.

[35] In the modified citrulline oxidase, further, the amino acid at the position corresponding to position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase; the amino acid at the position corresponding to position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid in the modified citrulline oxidase; the amino acid at the position corresponding to position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at the position corresponding to position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. The composition according to Embodiment 34, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine, and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

[36] A method for quantifying L-citrulline, comprising the step of contacting a sample that may contain L-citrulline with a modified citrulline oxidase according to any one of Embodiments 12 to 15.

[0016] This specification includes the disclosures of Japanese Patent Application No. 2025-055034, which forms the basis of the priority claim of this application.

[0017] As an effect of the present invention, the activity of peptidylarginine deiminase (PAD) contained in a sample can be measured. Furthermore, the activity of PAD4 contained in a sample can be measured. This can be used to diagnose or assist in the diagnosis of rheumatoid arthritis. Furthermore, this can be used to diagnose or assist in the diagnosis of fibrosis. Furthermore, this can be used to diagnose or assist in the diagnosis of ulcerative colitis. Furthermore, this can be used to diagnose or assist in the diagnosis of cancer. Furthermore, this can be used to diagnose or assist in the diagnosis of multiple sclerosis. Furthermore, this can be used to diagnose or assist in the diagnosis of Alzheimer's disease. Furthermore, the activity of PAD2 contained in a sample can be measured. Furthermore, this can be used to diagnose or assist in the diagnosis of PAD2-related diseases. Furthermore, this can be used to diagnose or assist in the diagnosis of Alzheimer's disease. Furthermore, this may enable the diagnosis of inflammatory bowel disease or assist in its diagnosis. Furthermore, this may enable the diagnosis of cancer or assist in its diagnosis. Furthermore, this may enable the diagnosis of inflammatory bowel disease or assist in its diagnosis.

[0018] This is a schematic diagram showing the procedure for measuring PAD activity. The results of comparing the absorbance changes at 555 nm for a PAD4 sample and a control sample without added PAD are shown. This is a plot of PAD4 concentration and absorbance. For the 554M mutant, the relationship between incubation time and absorbance change at 555 nm is shown. For the 554L mutant, the relationship between incubation time and absorbance change at 555 nm is shown. For the 554N mutant, the relationship between incubation time and absorbance change at 555 nm is shown. The time course of absorbance when peptide No. 4, peptide No. 9, or peptide No. 19 is added in the presence of 35 μg / mL PAD2 is shown. The time course of absorbance when peptide No. 4, peptide No. 9, or peptide No. 19 is added in the presence of 3.5 μg / mL PAD2 is shown. The reactivity with PAD2 and PAD4 when peptide No. 28 (MS{Cit}MG) is used as a substrate is shown. The reactivity with PAD2 and PAD4 when peptide No. 29 (KS{Cit}MG) is used as a substrate is shown. The reactivity with PAD2 and PAD4 when peptide No. 30 (HS{Cit}MG) is used as a substrate is shown. The change in absorbance over time when PAD2 is added when peptide No. 27 (KSRMG) is used as a substrate is shown. The change in absorbance over time when PAD2 is added when peptide No. 31 (KSRIG) is used as a substrate is shown. The change in absorbance over time when PAD2 is added when peptide No. 32 (KSRKG) is used as a substrate is shown. The change in absorbance over time when PAD2 is added when peptide No. 33 (KSRLG) is used as a substrate is shown. The change in absorbance over time when PAD2 is added when peptide No. 34 (KSRVG) is used as a substrate is shown. The change in absorbance over time when PAD2 or PAD4 is added when peptide No. 32 (KSRKG) is used as a substrate is shown. The following shows the change in absorbance over time when PAD2 or PAD4 is added using peptide No. 35 (KWRKG). The following shows the change in absorbance over time when PAD2 or PAD4 is added using peptide No. 36 (KIRKG).The following shows the time course of absorbance when peptide No. 37 (KPRKG) is used and PAD2 or PAD4 is added. The following shows the time course of absorbance when peptide No. 38 (KQRKG) is used and PAD2 or PAD4 is added. The following shows the time course of absorbance when peptide No. 35 (KWRKG) is used and PAD2 or PAD4 is added. The following shows the time course of absorbance when peptide No. 38 (KQRKG) is used and PAD2 or PAD4 is added. The following shows the time course of absorbance when MD{Cit}DG is used as a substrate, carboxypeptidase A is used, and dispase is present and absent. The following shows the time course of absorbance when MD{Cit}DG is used as a substrate, carboxypeptidase B is used, and dispase is present and absent. The following shows the time course of absorbance when MD{Cit}DG is used as a substrate, leucine aminopeptidase (LAP) is used, and dispase is present and absent.

[0019] In one embodiment, the present disclosure provides a method for measuring PAD activity in a sample. This method may include the steps of: (i) contacting a sample with a substrate peptide containing an L-arginine residue to allow the PAD contained in the sample to act on the substrate peptide containing the L-arginine residue (also referred to as the substrate peptide containing an L-arginine residue) to generate a peptide containing an L-citrulline residue; (ii) digesting the peptide containing the L-citrulline residue generated in step (i) using a peptidase to release L-citrulline; and (iii) oxidizing the L-citrulline released in step (ii) using a citrulline oxidase and measuring it.

[0020] In one embodiment, the substrate peptide containing the L-arginine residue may be a peptide having an amino acid sequence X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine], or a peptide consisting of the amino acid sequence. In this embodiment, PAD may be PAD4. In one embodiment, the substrate peptide containing the L-arginine residue may be a peptide consisting of the amino acid sequence Met-Asp-Arg-Asp-Gly. In another embodiment, the peptide may be a peptide consisting of Ser-Asp-Arg-Asp-Gly, Met-Gly-Arg-Asp-Gly, Ser-Gly-Arg-Asp-Gly, Met-Asp-Arg-Gly-Gly, Met-Gly-Arg-Gly-Gly, Ser-Asp-Arg-Gly-Gly, or Ser-Gly-Arg-Gly-Gly. In one embodiment, Phe-Gly-, Tyr-Gly-, or Gly- may be further added to the N-terminus of these peptides. In another embodiment, -Gly, -Gly-Gly, or -Gly-Tyr may be added to the C-terminus of these peptides. In one embodiment, Phe-Gly-, Tyr-Gly-, or Gly- may be added to the N-terminus of these peptides, and -Gly, -Gly-Gly, or -Gly-Tyr may be added to the C-terminus.

[0021] In another embodiment, PAD is PAD2, and the substrate peptide containing the L-arginine residue has an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; and the third amino acid residue is L-arginine (Arg). The fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, and the fifth amino acid residue X5 is glycine. This may be a peptide having this amino acid sequence, or a peptide consisting of this amino acid sequence. In one embodiment, the substrate peptide containing the L-arginine residue may be a peptide consisting of Lys-Gln-Arg-Lys-Gly. In another embodiment, the peptide may be a peptide selected from the group consisting of Met-Ser-Arg-Met-Gly, Asp-Ser-Arg-Met-Gly, Glu-Ser-Arg-Met-Gly, His-Ser-Arg-Met-Gly, Lys-Ser-Arg-Met-Gly, Lys-Ser-Arg-Ile-Gly, Lys-Ser-Arg-Lys-Gly, Lys-Ser-Arg-Leu-Gly, Lys-Ser-Arg-Val-Gly, Lys-Trp-Arg-Lys-Gly, and Lys-Ile-Arg-Lys-Gly.

[0022] The peptidase is not particularly limited as long as it can release L-citrulline from a peptide containing an L-citrulline residue. The peptidase only needs to recognize a peptide containing an L-citrulline residue as a substrate, and even enzymes classified as proteases are included in the definition of peptidase as used herein for convenience. In some embodiments, the peptidase may be, but is not limited to, porcine pancreas-derived carboxypeptidase B, recombinant rat carboxypeptidase B / CPB protein, carboxypeptidase Y, Pfu aminopeptidase I, Streptomyces glyceus-derived aminopeptidase I, microsomal porcine kidney-derived leucine aminopeptidase, recombinant human methionine aminopeptidase 1 / METAP1 protein, recombinant Escherichia coli-derived methionine aminopeptidase 1 / METAP1 His protein, recombinant Aeromonas-derived aminopeptidase, recombinant mouse-derived aminopeptidase P2 / XPNPEP2 protein (carrier-free), recombinant human-derived aminopeptidase LRAP / ERAP2 protein (carrier-free), or a combination thereof. In some embodiments, the peptidase may be endopeptidase or exopeptidase. Examples of exopeptidases include, but are not limited to, aminopeptidases, such as leucine aminopeptidase, and carboxypeptidases, such as carboxypeptidase A and carboxypeptidase B. Examples of endopeptidases include, but are not limited to, dispase. In one embodiment, multiple types of peptidases may be used to release L-citrulline from a peptide containing an L-citrulline residue. In one embodiment, the first endopeptidase can be combined with the second endopeptidase. In one embodiment, the endopeptidase can be combined with the exopeptidase. In one embodiment, the first exopeptidase can be combined with the second exopeptidase. Three or more types of peptidases can also be combined as subcombinations of these.In one embodiment, the peptidase may be a combination of a carboxypeptidase (e.g., carboxypeptidase A or carboxypeptidase B) and an endopeptidase (e.g., dispase). In another embodiment, the peptidase may be a combination of an aminopeptidase (e.g., leucine aminopeptidase) and an endopeptidase (e.g., dispase).

[0023] In one embodiment, the oxidation of L-citrulline by citrulline oxidase can be measured by a colorimetric assay using peroxidase / hydrogen peroxide. When citrulline oxidase acts on L-citrulline, the L-citrulline is oxidized, and the oxygen molecule accepts electrons and is converted into hydrogen peroxide. The generated hydrogen peroxide can be detected by peroxidase and a chromogenic substrate. Detection can be performed at a wavelength corresponding to the absorption wavelength of the chromogenic substrate. For example, the color produced by the oxidative condensation reaction between 4-aminoantipyrine (4-AA) and TOOS has a maximum absorption wavelength at 555 nm, so when these are used as chromogenic substrates, the absorbance at 555 nm can be measured. Alternatively, when a leuco-type dye (leucomethylene blue derivative) is used, it changes to a blue dye through oxidation. In this case, the maximum absorption wavelength is around 665 nm, so when a leuco-type dye is used as a chromogenic dye, the absorbance at 665 nm can be measured. Examples of leuco-type dyes include, but are not limited to, DA-67, KN-301, and KN-302. The chromogenic substrate, chromogenic dye, and leuco-type dye can be commercially available or synthesized. Those skilled in the art can synthesize known chromogenic substrates, chromogenic dyes, and leuco-type dyes using organic synthesis techniques.

[0024] In one embodiment, the present disclosure provides a method for diagnosing rheumatoid arthritis or a method for assisting in the diagnosis of rheumatoid arthritis. In this method, the sample is a biological sample obtained from a subject, and the PAD activity, for example, PAD4 activity, of the biological sample is measured by the method of the present disclosure. If the PAD activity, for example, PAD4 activity, is higher than a threshold, the subject can be diagnosed with rheumatoid arthritis, or a diagnosis can be assisted in this diagnosis. The threshold can be appropriately set from the mean or median PAD activity, for example, PAD4 activity, of healthy individuals and the mean or median PAD activity, for example, PAD4 activity, of rheumatoid arthritis patients. If the PAD activity, for example, PAD4 activity, of a sample is higher than a threshold, the subject from whom the sample was obtained can be diagnosed with rheumatoid arthritis, or a diagnosis can be assisted in this diagnosis. If the subject is diagnosed with rheumatoid arthritis, treatment with known rheumatoid arthritis drugs (e.g., methotrexate, TNFα inhibitors, etc.) can be administered.

[0025] In one embodiment, the present disclosure provides a method for diagnosing fibrosis or a method to assist in the diagnosis of fibrosis. In this method, the sample is a biological sample obtained from a subject, and the PAD4 activity of the biological sample is measured by the method of the present disclosure. If the PAD4 activity is higher than a threshold, the subject can be diagnosed with fibrosis, or a diagnosis can be assisted in the diagnosis. The threshold can be appropriately set from the mean or median PAD4 activity of healthy individuals and the mean or median PAD4 activity of fibrosis patients. If the PAD4 activity of a sample is higher than a threshold, the subject from whom the sample was obtained can be diagnosed with fibrosis, or a diagnosis can be assisted in the diagnosis. If the subject is diagnosed with fibrosis, treatment with known fibrosis treatment drugs can be administered.

[0026] In one embodiment, the Disclosure provides a method for diagnosing cancer or a method for assisting in the diagnosis of cancer. In this method, the sample is a biological sample obtained from a subject, and the PAD activity, for example, PAD4 activity or PAD2 activity, of the biological sample is measured by the method of the Disclosure. If the PAD4 activity or PAD2 activity is higher than a threshold, the subject can be diagnosed with cancer, or a diagnosis can be assisted in such diagnosis. The threshold can be appropriately set from the mean or median PAD4 activity or PAD2 activity of healthy individuals and the mean or median PAD4 activity or PAD2 activity of cancer patients. If the PAD4 activity or PAD2 activity of a sample is higher than a threshold, the subject from whom the sample was obtained can be diagnosed with cancer, or a diagnosis can be assisted in such diagnosis. If the subject is diagnosed with cancer, treatment with a known cancer drug (or its equivalent, for example, a cancer drug developed after the filing of this application) can be administered. In one embodiment, the cancer may be a cancer with increased PAD4 activity. In one embodiment, the cancer may be a cancer with increased PAD2 activity.

[0027] In one embodiment, the present disclosure provides a method for diagnosing multiple sclerosis, or a method to assist in the diagnosis of multiple sclerosis. In this method, the sample is a biological sample obtained from a subject, and the PAD activity, for example, PAD4 activity or PAD2 activity, of the biological sample is measured by the method of the present disclosure. If the PAD activity, for example, PAD4 activity or PAD2 activity, is higher than a threshold, the subject can be diagnosed, or a diagnosis can be assisted in, that has multiple sclerosis. The threshold can be appropriately set from the mean or median PAD activity of healthy individuals, for example, PAD4 activity or PAD2 activity, and the mean or median PAD activity of patients with multiple sclerosis, for example, PAD4 activity or PAD2 activity. If the PAD activity, for example, PAD4 activity or PAD2 activity, of a sample is higher than a threshold, the subject from whom the sample was obtained can be diagnosed, or a diagnosis can be assisted in, that has multiple sclerosis. If a subject is diagnosed with multiple sclerosis, treatment may be administered using a known multiple sclerosis drug (or an equivalent thereof, for example, a multiple sclerosis drug developed after the filing of this application). In one embodiment, multiple sclerosis may be characterized by increased PAD4 activity. In another embodiment, multiple sclerosis may be characterized by increased PAD2 activity.

[0028] In one embodiment, the present disclosure provides a method for diagnosing ulcerative colitis, or a method to assist in the diagnosis of ulcerative colitis. In this method, the sample is a biological sample obtained from a subject, and the PAD4 activity of the biological sample is measured by the method of the present disclosure. If the PAD4 activity is higher than a threshold, the subject can be diagnosed, or a diagnosis can be assisted in, that has ulcerative colitis. The threshold can be appropriately set from the mean or median PAD4 activity of healthy individuals and the mean or median PAD4 activity of patients with ulcerative colitis. If the PAD4 activity of a sample is higher than a threshold, the subject from which the sample was obtained can be diagnosed, or a diagnosis can be assisted in, that has ulcerative colitis. If the subject is diagnosed with ulcerative colitis, treatment can be administered using a known ulcerative colitis drug (or its equivalent, for example, an ulcerative colitis drug developed after the filing of this application).

[0029] In one embodiment, the present disclosure provides a method for diagnosing Alzheimer's disease or a method to assist in the diagnosis of Alzheimer's disease. In this method, the sample is a biological sample obtained from a subject, and the PAD activity, for example, PAD4 activity or PAD2 activity, of the biological sample is measured by the method of the present disclosure. If the PAD activity, for example, PAD4 activity or PAD2 activity, is higher than a threshold, the subject can be diagnosed, or a diagnosis can be assisted in, that has Alzheimer's disease. The threshold can be appropriately set from the mean or median PAD activity of healthy individuals, for example, PAD4 activity or PAD2 activity, and the mean or median PAD activity of patients with Alzheimer's disease, for example, PAD4 activity or PAD2 activity. If the PAD activity, for example, PAD4 activity or PAD2 activity, of a sample is higher than a threshold, the subject from whom the sample was obtained can be diagnosed, or a diagnosis can be assisted in, that has Alzheimer's disease. If a subject is diagnosed with Alzheimer's disease, treatment may be administered using a known Alzheimer's disease drug (or an equivalent thereof, such as an Alzheimer's disease drug developed after the filing of this application). In one embodiment, Alzheimer's disease may be characterized by increased PAD4 activity. In another embodiment, Alzheimer's disease may be characterized by increased PAD2 activity.

[0030] In one embodiment, the present disclosure provides a method for diagnosing inflammatory bowel disease, or a method for assisting in the diagnosis of inflammatory bowel disease. In this method, the sample is a biological sample obtained from a subject, and the PAD2 activity of the biological sample is measured by the method of the present disclosure. If the PAD2 activity is higher than a threshold, the subject can be diagnosed, or a diagnosis can be assisted in, that has inflammatory bowel disease. The threshold can be appropriately set from the mean or median PAD2 activity of healthy individuals and the mean or median PAD2 activity of patients with inflammatory bowel disease. If the PAD2 activity of a sample is higher than a threshold, the subject from which the sample was obtained can be diagnosed, or a diagnosis can be assisted in, that has inflammatory bowel disease. If the subject is diagnosed with inflammatory bowel disease, treatment can be administered using a known inflammatory bowel disease drug (or its equivalent, for example, an inflammatory bowel disease drug developed after the filing of this application).

[0031] In one embodiment, the citrulline oxidase may be one described in International Publication No. 2021 / 054375. In another embodiment, the citrulline oxidase may be a modified citrulline oxidase of the present disclosure. In one embodiment, the modified citrulline oxidase of the present disclosure may be based on the original polypeptide. In one embodiment, the original polypeptide may have, for example, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, for example 100% amino acid sequence identity with SEQ ID NO: 1. In another embodiment, the unmodified polypeptide may have one or more amino acids substituted, deleted, added, and / or inserted at positions other than, for example, positions 291, 302, 221, and 554 of SEQ ID NO: 1, and positions 502, 479, 305, 310, and 113. Here, one or more amino acids means 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 4, for example 1 to 3, for example 1 or 2 amino acids. In one embodiment, the unmodified polypeptide may be a polypeptide derived from the genus Streptomyces. In one embodiment, the unmodified polypeptide may have FAD as a coenzyme. Also, the modified citrulline oxidase may have FAD as a coenzyme.

[0032] In one embodiment, the modified citrulline oxidase has a glutamine substitution at the amino acid corresponding to position 291 of SEQ ID NO: 1. In one embodiment, the modified citrulline oxidase (e.g., the 291Q mutant) may have reduced reactivity to L-arginine and increased activity to L-citrulline compared to the original polypeptide.

[0033] In one embodiment, the modified citrulline oxidase (e.g., the 291Q mutant) exhibits a decrease in activity (assuming 100%) toward L-arginine compared to the original polypeptide, with the modified citrulline oxidase being reduced by 10% or more (i.e., activity below 90%), 20% or more (i.e., activity below 80%), 30% or more (i.e., activity below 70%), 40% or more (i.e., activity below 60%), 50% or more (i.e., activity below 50%), 60% or more (i.e., activity below 40%), 70% or more (i.e., activity below 30%), and 80% or more (i.e., activity below 20%). The activity level may be 0%, or 90% or more lower (i.e., less than 10% activity), and the activity for L-citrulline (assuming 100%) may be increased by 10% or more (i.e., 110%), 20% or more (i.e., 120%), 30% or more (i.e., 130%), 40% or more (i.e., 140%), 50% or more (i.e., 150%), 60% or more (i.e., 160%), 70% or more (i.e., 170%), 80% or more (i.e., 180%), 90% or more (i.e., 190%), or 100% or more (i.e., 200%). If no activity toward L-citrulline is detected in the original polypeptide, but activity toward L-citrulline is detected in the modified citrulline oxidase, for convenience, this will be included in cases where the activity toward L-citrulline (100%) increases by 10% or more (i.e., to 110%), or increases by 100% or more (i.e., to 200%). Note that the 291Q variant referred to here only requires that the amino acid at position 291 of Sequence ID No. 1 be substituted with glutamine; it does not mean that there are no amino acid substitutions at other positions.

[0034] In one embodiment, the modified citrulline oxidase of this disclosure not only has the amino acid at position 291 of SEQ ID NO: 1 substituted with glutamine, but may also have the amino acid at position 302 of SEQ ID NO: 1 substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase, the amino acid at position 554 of SEQ ID NO: 1 substituted with methionine, leucine, or asparagine, or isoleucine, or glutamine in the modified citrulline oxidase, and / or the amino acid at position 221 of SEQ ID NO: 1 substituted with aspartic acid or glutamic acid in the modified citrulline oxidase. The inventors have confirmed that mutants of the polypeptide with the amino acid sequence of SEQ ID NO: 1 having 291Q and 302I mutations have citrulline oxidase activity. Therefore, there is a high probability that a polypeptide having citrulline oxidase activity can also be obtained by substituting the position corresponding to position 302 of SEQ ID NO: 1 with leucine, which has a structure and properties similar to isoleucine. Similarly, the inventors confirmed that a mutant of the polypeptide with the amino acid sequence of SEQ ID NO: 1, having the 291Q / 302M / 221D mutation, possesses citrulline oxidase activity. Therefore, there is a high probability that a polypeptide possessing citrulline oxidase activity can be obtained by substituting the position corresponding to position 221 of SEQ ID NO: 1 with glutamic acid, which is structurally and propertyly similar to aspartic acid. Similarly, the inventors confirmed that mutants of the polypeptide with the amino acid sequence of SEQ ID NO: 1, having the 291Q / 302M / 221D / 554L mutation and the 291Q / 302M / 221D / 554N mutation, possess citrulline oxidase activity. Therefore, there is a high probability that a polypeptide possessing citrulline oxidase activity can be obtained by substituting the position corresponding to position 554 of SEQ ID NO: 1 with isoleucine, which is structurally and propertyly similar to leucine, and also a high probability that a polypeptide possessing citrulline oxidase activity can be obtained by substituting it with glutamine, which is structurally and propertyly similar to asparagine.

[0035] In one embodiment, in the modified citrulline oxidase of the present disclosure, the amino acid at the position corresponding to position 502 of SEQ ID NO: 1 may be further substituted with glycine or alanine in the modified citrulline oxidase, the amino acid at the position corresponding to position 479 of SEQ ID NO: 1 may be substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine or threonine in the modified citrulline oxidase, the amino acid at the position corresponding to position 305 of SEQ ID NO: 1 may be substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine or tryptophan in the modified citrulline oxidase, the amino acid at the position corresponding to position 310 of SEQ ID NO: 1 may be substituted with valine, leucine or methionine in the modified citrulline oxidase, and / or the amino acid at the position corresponding to position 113 of SEQ ID NO: 1 may be substituted with arginine in the modified citrulline oxidase.

[0036] In one embodiment, the modified citrulline oxidase of the present disclosure may have 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, for example 100%, provided that it has one or more amino acid substitutions at positions corresponding to positions 291, 302, 554, or 221 of SEQ ID NO: 37, or positions 502, 479, 305, 310, or 113 of SEQ ID NO: 37. Furthermore, in a modified citrulline oxidase having a predetermined amino acid sequence identity with SEQ ID NO: 37, the amino acid at position 291 of SEQ ID NO: 1 may be glutamine, the amino acid at position 302 of SEQ ID NO: 1 may be methionine, isoleucine, or leucine, the amino acid at position 554 of SEQ ID NO: 1 may be methionine, leucine, asparagine, isoleucine, or glutamine, and / or the amino acid at position 221 of SEQ ID NO: 1 may be aspartic acid or glutamic acid, and / or the amino acid at position 502 of SEQ ID NO: 1 may be glycine or ara It may also be nin, and the amino acid at position 479 of SEQ ID NO: 1 may be leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine; the amino acid at position 305 of SEQ ID NO: 1 may be valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan; the amino acid at position 310 of SEQ ID NO: 1 may be valine, leucine, or methionine; and / or the amino acid at position 113 of SEQ ID NO: 1 may be arginine. In another embodiment, the modified citrulline oxidase of the present disclosure may be, for example, obtained by substituting, deleting, adding, and / or inserting one or more amino acids at positions other than 291, 302, 221, and 554, and 502, 479, 305, 310, and 113 of SEQ ID NO: 37.

[0037] In one embodiment, the Disclosure provides the modified citrulline oxidase described above. In one embodiment, the Disclosure provides a composition, reagent, and / or kit for PAD activity measurement comprising the modified citrulline oxidase described above. In one embodiment, the composition, reagent, and / or kit for PAD activity measurement (e.g., a composition for PAD2 activity measurement, or a composition for PAD4 activity measurement) may further comprise a substrate peptide comprising the L-arginine residue described above. In one embodiment, the composition, reagent, and / or kit for PAD activity measurement may further comprise a peptidase. The peptidase may recognize the peptide comprising the L-citrulline residue described above as a substrate.

[0038] In one embodiment, the Disclosure provides a polynucleotide encoding the modified citrulline oxidase described above, a vector containing the polynucleotide, and a host cell containing the vector. In one embodiment, the Disclosure provides a method for producing the modified citrulline oxidase. This method may include the steps of culturing the host cell to produce the modified citrulline oxidase of the Disclosure, and obtaining the produced modified citrulline oxidase.

[0039] In one embodiment, the Disclosure provides a composition, reagent, and / or kit for measuring PAD activity, comprising a substrate peptide containing the above-described L-arginine residue. In one embodiment, PAD is PAD4. In another embodiment, PAD is PAD2. In one embodiment, this composition, reagent, and / or kit for measuring PAD activity is for measuring PAD activity by an enzymatic method. In one embodiment, this composition, reagent, and / or kit for measuring PAD activity excludes methods other than enzymatic methods, such as mass spectrometry, chromatography, or HPLC for measuring PAD activity. In one embodiment, the Disclosure provides a composition, reagent, and / or kit for diagnosing rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, comprising the above-described composition, reagent, and / or kit for measuring PAD4 activity. This composition, reagent, and / or kit for diagnosing rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease may include the modified citrulline oxidase of the Disclosure. In one embodiment, the composition, reagent, and / or kit for the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease is for measuring PAD activity, more specifically PAD4 activity, by an enzymatic method. In one embodiment, the composition, reagent, and / or kit for the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease excludes methods other than enzymatic methods, such as methods for measuring PAD4 activity by mass spectrometry, chromatography, or HPLC. In another embodiment, the Disclosure provides a composition, reagent, and / or kit for the diagnosis of Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, comprising the composition, reagent, and / or kit for measuring PAD2 activity described above. The composition, reagent, and / or kit for the diagnosis of Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease may include the modified citrulline oxidase of the Disclosure. In one embodiment, the composition, reagent, and / or kit for the diagnosis of Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease is for measuring PAD activity, more specifically PAD2 activity, by enzymatic method.In one embodiment, methods for measuring PAD2 activity by methods other than enzymatic methods, e.g., mass spectrometry, chromatography, HPLC, are excluded from the composition, reagent, and / or kit for diagnosing Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease.

[0040] The modified citrulline oxidase of the present disclosure is based on the GLOD of SEQ ID NO: 1. GLOD uses flavin adenine dinucleotide (FAD) as a coenzyme. Oxidoreductases that use FAD as a coenzyme are known to have an FAD-binding motif sequence, for example, the Gly-Xaa-Gly-Xaa-Xaa-Gly motif (wherein Xaa is any amino acid). For example, when SEQ ID NO: 1 is used as the reference sequence, the amino acid sequence "Gly-Ala-Gly-Ile-Ala-Gly" at positions 51 to 56 of SEQ ID NO: 1 corresponds to the FAD-binding motif sequence Gly-Xaa-Gly-Xaa-Xaa-Gly. In one embodiment, with respect to the variants of the present disclosure, based on SEQ ID NO: 1, Gly at the positions corresponding to positions 51, 53, and 56, respectively, does not need to undergo amino acid substitution. In one embodiment, Gly at positions 51, 53, and 56 of L-glutamate oxidase SEQ ID NO: 1 is not subjected to amino acid substitution.

[0041] (Reference Sequence) For convenience in the present specification, SEQ ID NO: 1 is used as the reference sequence to define each position of the polypeptide. SEQ ID NO: 1 is a polypeptide based on glutamate oxidase (M7GLOD) derived from Streptomyces sp. MOE-7. However, the N-terminal secretion signal sequence (MTTDTARRHTGAER) in the native sequence has been removed.

[0042] When a variant based on SEQ ID NO: 1 was prepared, L-citrulline oxidation activity was confirmed. A person skilled in the art can prepare similar variants based on the disclosure herein and confirm the presence or absence of the activity through routine testing. In the present specification, variants that do not have L-citrulline oxidation activity are excluded from the modified citrulline oxidase of the present disclosure.

[0043] GLODs are widely distributed in nature and can be obtained by searching for enzymes of microorganism, animal, or plant origin. Microorganisms, for example, can be obtained from actinomycetes, filamentous fungi, yeasts, or bacteria. In this specification, the origin of GLOD is not particularly limited and means GLOD derived from microorganisms of the genera Streptomyces, e.g., Streptomyces sp. X-119-6, and Streptomyces sp. MOE7, Streptomyces lydicus, Azotobacter, Embleya, Kitasatospora, Saccharothrix, Alloactinosynnema, Streptoalloteichus, Actinoalloteichus, Catenulisporora, Nannocystis, Actinobacteria, Actinophytocola, Spharisporangium, Microbispora, Streptosporangium, Phytohabitans, Haliangium, Archangium, Streptacidiphilus, Saccharothrix, or Trichoderma, and unless otherwise specified, includes both wild types and their variants.

[0044] (Obtaining the gene encoding GLOD) To obtain the gene encoding GLOD (hereinafter also simply referred to as the "GLOD gene"), commonly used gene cloning methods are employed. For example, chromosomal DNA or mRNA can be extracted from GLOD-producing microorganisms or various cells by conventional methods. Furthermore, cDNA can be synthesized using the mRNA as a template. A library of chromosomal DNA or cDNA can be prepared using the chromosomal DNA or cDNA obtained in this way.

[0045] Next, based on the amino acid sequence of GLOD, a suitable probe DNA can be synthesized and used to select the GLOD gene from a library of chromosomal DNA or cDNA. Alternatively, a suitable primer DNA can be prepared based on the amino acid sequence, and the DNA containing the target gene fragment encoding GLOD can be amplified by a suitable polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method. These DNA fragments can then be ligated to obtain DNA containing the full length of the target GLOD gene.

[0046] Examples of GLOD genes include, but are not limited to, the GLOD gene derived from Streptomyces sp. X-119-6, the GLOD gene derived from Streptomyces sp. MOE7, and the GLOD gene derived from Streptomyces lydicus.

[0047] The GLOD gene or citrulline oxidase gene may be ligated to a vector. Examples of vectors include plasmids, bacteriophages, and cosmids; for example, pBluescriptII SK+ (Stratagene). Plasmids can be obtained by conventional methods. For example, the GLOD gene-containing plasmid can be extracted and purified using the GenElute Plasmid Miniprep Kit (Sigma-Aldrich). The obtained GLOD gene can be manipulated to create GLOD mutant genes or obtain purified enzymes.

[0048] (Mutation of the GLOD gene or citrulline oxidase gene) Mutation of the GLOD gene or citrulline oxidase gene can be carried out by any known method depending on the desired mutation. Specifically, methods such as contacting and acting on the GLOD gene or citrulline oxidase gene, or recombinant DNA incorporating the gene, with a mutagenic drug; ultraviolet irradiation; genetic engineering techniques; or protein engineering techniques can be widely used.

[0049] Examples of mutagenic drugs used in the above-mentioned mutagenesis treatment include hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrite, sulfite, hydrazine, formic acid, or 5-bromouracil.

[0050] The conditions for this contact and action can be adjusted according to the type of drug used, and are not particularly limited as long as the desired mutation can be induced in the GLOD gene or citrulline oxidase gene. Typically, the desired mutation can be induced by contact and action for 10 minutes or more, preferably 10 to 180 minutes, at a drug concentration of 0.5 to 12 M and a reaction temperature of 20 to 80°C. Even when ultraviolet irradiation is performed, it can be carried out according to the conventional method as described above.

[0051] As a method utilizing protein engineering techniques, techniques generally known as site-specific mutagenesis can be used. Examples include the Kramer method (Nucleic Acids Res., 12, 9441-9456 (1984)), the Eckstein method (Nucleic Acids Res., 13, 8749-8764 (1985): Nucleic Acids Res., 13, 8765 (1985): Nucleic Acids Res, 14, 9679 (1986)), and the Kunkel method (Proc. Natl. Acid. Sci. USA, 82, 488-492 (1985)).

[0052] In addition to the gene modification methods described above, it is also possible to directly synthesize the desired GLOD gene or citrulline oxidase gene using organic synthesis or enzymatic synthesis methods.

[0053] The nucleotide sequence of the GLOD gene or citrulline oxidase gene can be confirmed, for example, using a multi-capillary DNA analysis system such as the Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific).

[0054] (Transformation and Transduction) The GLOD gene or citrulline oxidase gene can be incorporated into vectors such as bacteriophages, cosmids, or plasmids used for the transformation of prokaryotic or eukaryotic cells by conventional methods. Using these vectors, the host corresponding to each vector can be transformed or transduced by conventional methods.

[0055] In one embodiment, GLOD or citrulline oxidase can be expressed using prokaryotic cells, such as microorganisms of the genus Escherichia, such as Escherichia coli; microorganisms of the genus Brevibacillus, such as Brevibacillus choshinensis; microorganisms of the genus Corynebacterium, such as Corynebacterium glutamicum; and microorganisms of the genus Streptomyces, such as Streptomyces violaceoruber. Examples of Escherichia coli hosts include, but are not limited to, various strains of Escherichia coli, such as strain K-12, strain JM109, strain DH5α, strain BL21, strain JM109(DE3), strain DH5α(DE3), strain BL21(DE3), strain TG1, strain 1100, strain W3110, strain C600, etc. The host is transformed or transduced to obtain host cells (transformed organisms) into which the GLOD gene or citrulline oxidase gene has been introduced. Methods for transferring the recombinant vector into these host cells include, for example, transferring the recombinant DNA in the presence of calcium ions when the host cell is a microorganism belonging to Escherichia korye, or using electroporation. Furthermore, commercially available competent cells (e.g., ECOS Competent Escherichia korye BL21(DE3); manufactured by Nippon Gene) may be used. The GLOD gene may be codon-optimized according to the host it expresses.

[0056] In one embodiment, GLOD or citrulline oxidase can be expressed using eukaryotic cells. An example of a eukaryotic host cell is yeast. Examples of microorganisms classified as yeast include yeasts belonging to the genera Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, Pichia, and Candida. The inserted gene may include a marker gene to enable the selection of transformed cells. Examples of marker genes include genes that complement the host's nutritional requirements, such as URA3 and TRP1. Furthermore, it is desirable that the inserted gene includes a promoter or other regulatory sequence (e.g., enhancer sequence, terminator sequence, polyadenylation sequence, etc.) that can express the target gene in the host cell. Specific examples of promoters include the GAL1 promoter and the ADH1 promoter. As a method for transforming yeast, known methods such as the lithium acetate method (Methods Mol. Cell. Biol., 5, 255-269 (1995)) and electroporation (J Microbiol Methods 55 (2003) 481-484) can be suitably used, but are not limited to these. Transformation can be carried out using any method, including the spheroplast method and the glass bead method.

[0057] Furthermore, other examples of eukaryotic host cells include fungal cells (including filamentous fungi) such as those of the genera Aspergillus and Trichoderma. The method for producing fungal cell transformants is not particularly limited, and one example is to insert a gene encoding GLOD or citrulline oxidase into a host filamentous fungus in a manner that expresses it, according to a conventional method. Specifically, a DNA construct is prepared by inserting the gene encoding GLOD between an expression-inducing promoter and a terminator, and then the host filamentous fungus is transformed with the DNA construct containing the gene encoding GLOD or citrulline oxidase to obtain a transformant that overexpresses the gene encoding GLOD or citrulline oxidase.

[0058] The method of inserting a gene encoding GLOD or citrulline oxidase into a host filamentous fungus in a manner in which it is expressed is not particularly limited, but examples include methods of directly inserting it onto the chromosome of a host organism using homologous recombination, and methods of introducing it into a host filamentous fungus by ligating it onto a plasmid vector.

[0059] In methods utilizing homologous recombination, a DNA construct can be ligated between sequences homologous to the upstream and downstream regions of a recombination site on a chromosome and inserted into the genome of the host filamentous fungus. By overexpressing this construct within the host filamentous fungus under the control of its own high-expression promoter, a transformant can be obtained through self-cloning. The high-expression promoter is not particularly limited, but examples include the promoter region of the translation elongation factor TEF1 gene (tef1), the promoter region of the α-amylase gene (amy), and the promoter region of the alkaline protease gene (alp).

[0060] In the vector-based method, a DNA construct can be incorporated into a plasmid vector used for filamentous fungal transformation using a standard method, and the corresponding host filamentous fungus can then be transformed using a standard method.

[0061] Such suitable vector-host systems are not particularly limited as long as they are capable of producing GLOD or citrulline oxidase in the host filamentous fungus, and examples include the pUC19 and filamentous fungus system, and the pSTA14 (Mol. Gen. Genet. 218, 99-104, 1989) and filamentous fungus system.

[0062] While it is preferable to introduce the DNA construct into the chromosome of the host filamentous fungus, another method is to incorporate the DNA construct into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)), thereby allowing it to be used without being introduced into the chromosome.

[0063] The DNA construct may include marker genes to enable the selection of transformed cells. The marker genes are not particularly limited and include, for example, genes that complement the host's nutritional requirements, such as pyrG, niaD, and adeA; and drug resistance genes to drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also preferably includes promoters, terminators, and other regulatory sequences (e.g., enhancers, polyadenylation sequences) that enable the overexpression of genes encoding GLOD or citrulline oxidase in host cells. The promoters are not particularly limited but include appropriate inductive and constitutive promoters, such as the tef1 promoter, alp promoter, and amy promoter. The terminators are also not particularly limited but include, for example, the alp terminator, amy terminator, and tef1 terminator.

[0064] In a DNA construct, the expression regulatory sequence of the gene encoding GLOD or citrulline oxidase is not necessarily required if the DNA fragment containing the gene encoding GLOD or citrulline oxidase to be inserted contains a sequence with expression regulatory function. Furthermore, when transformation is performed by co-transformation, the DNA construct may not need to contain a marker gene.

[0065] One embodiment of the DNA construct is, for example, a DNA construct in which the tef1 gene promoter, the gene encoding GLOD, the alp gene terminator, and the pyrG marker gene are linked to the In-Fusion Cloning Site located at the multi-cloning site of pUC19.

[0066] As a method for transforming filamentous fungi, a method known to those skilled in the art can be appropriately selected. For example, after preparing protoplasts of the host filamentous fungus, the protoplast PEG method using polyethylene glycol and calcium chloride can be used (see, for example, Mol. Gen. Genet. 218, 99-104, 1989, Japanese Patent Publication No. 2007-222055). The culture medium for regenerating the transformed filamentous fungus should be appropriate depending on the host filamentous fungus and the transformation marker gene used. For example, if Aspergillus soybean is used as the host filamentous fungus and the pyrG gene is used as the transformation marker gene, the regeneration of the transformed filamentous fungus can be carried out, for example, in Czapek-Dox minimal medium (DIFCO) containing 0.5% agar and 1.2 M sorbitol.

[0067] Furthermore, for example, in order to obtain the transformed filamentous fungus of the present invention, homologous recombination may be used to replace the promoter of the gene encoding GLOD or citrulline oxidase, which is originally present on the chromosome of the host filamentous fungus, with a high-expression promoter such as tef1. In this case as well, it is preferable to insert a transformation marker gene such as pyrG in addition to the high-expression promoter. For example, for this purpose, referring to Example 1 and Figure 1 described in Japanese Patent Application Publication No. 2011-239681, a transformation cassette consisting of the upstream region of the gene encoding GLOD or citrulline oxidase - transformation marker gene - high-expression promoter - all or part of the gene encoding GLOD or citrulline oxidase can be used. In this case, the upstream region of the gene encoding GLOD or citrulline oxidase and all or part of the gene encoding GLOD or citrulline oxidase are used for homologous recombination. All or part of the gene encoding GLOD or citrulline oxidase can include the region from the start codon to the middle. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

[0068] The fact that the transformed filamentous fungus of the present invention has been produced can be confirmed by culturing the transformed filamentous fungus of the present invention under conditions in which enzymatic activity of GLOD or citrulline oxidase is observed, and then confirming the activity of GLOD or citrulline oxidase in the culture obtained after culturing.

[0069] Furthermore, confirmation that the transformed filamentous fungus of the present invention has been produced may be performed by extracting chromosomal DNA from the transformed filamentous fungus, performing PCR using this as a template, and confirming that a PCR product capable of amplification is produced when transformation occurs.

[0070] For example, PCR is performed using a combination of a forward primer for the promoter sequence and a reverse primer for the transformation marker gene sequence to confirm that a product of the expected length is produced.

[0071] The host may be a known microorganism, a known strain, or an equivalent or similar of a known microorganism or strain described herein. An equivalent is a host that exhibits equivalent function with respect to the recombinant expression of proteins. Equivalents include hosts created and modified based on hosts that were known at the time of filing of this application, which were developed after the filing of this application, and hosts that are similar in nature to hosts that were known at the time of filing of this application and were discovered after the filing of this application. With regard to the scientific name or classification of a microorganism, if there is a change in the scientific name, genus name, classification, etc. after the filing of this application, the description in this specification shall take precedence, and the time of filing of this application shall be the basis.

[0072] (High-throughput screening) Citrulline oxidase can also be subjected to high-throughput screening to obtain functional citrulline oxidase variants. For example, a library of transformed or transduced strains containing the mutant citrulline oxidase gene may be prepared and subjected to high-throughput screening based on microtiter plates, or to ultra-high-throughput screening based on droplet microfluidics. An example is a method of constructing a combinatorial library of variant-encoding mutant genes and then screening a large population of mutant GLODs using phage display (e.g., Chem. Rev. 105 (11): 4056-72, 2005), yeast display (e.g., Comb Chem High Throughput Screen. 2008;11(2): 127-34), or bacterial display (e.g., Curr Opin Struct Biol 17: 474-80, 2007). See also Agresti et al, "Ultrahigh-throughput screening in drop-based microfluidics for directed evolution," Proceedings of the National Academy of Sciences 107 (9): 4004-4009 (Mar, 2010). The description of ultrahigh-throughput screening methods that can be used for screening GLOD variants from that document is incorporated herein by reference. For example, a library can be constructed by error-prone PCR. Alternatively, a library may be constructed by introducing mutations targeting the regions and locations described herein or their corresponding regions and locations using saturated mutagenesis. Using the library, suitable cells such as electrocompetent EBY-100 cells can be transformed to obtain approximately 10^7 variants (10 million). Yeast cells transformed with the library can then be subjected to cell sorting. Polydimethoxysiloxane (PDMS) microfluidic devices prepared using standard soft lithography may also be used.Monodisperse droplets can be formed using a flow focus device. The formed droplets containing individual mutants can be subjected to a suitable sorting device. When selecting cells, the presence or absence of citrulline oxidase activity can be utilized. For example, a reaction solution with a composition that develops color when citrulline oxidase acts on it may be used. For example, when using a dye produced by condensing 4-aminoantipyrine (4-AA) and TOOS with peroxidase, the absorbance at 555 nm may be measured using a 96-well plate, 192-well plate, 384-well plate, 9600-well plate, etc., and a plate reader. Mutation introduction and selection may be repeated multiple times. Mutation here includes amino acid substitution, insertion, deletion, and / or addition.

[0073] For example, 1 to 10 mutations can be introduced into citrulline oxidase, and citrulline oxidase activity can be confirmed. Then, starting from the citrulline oxidase mutant confirmed to have activity, another 1 to 10 mutations can be introduced, and activity can be confirmed. A series of high-throughput screenings (for example, the above method of obtaining and screening approximately 10^7 mutants) can be repeated for 2 or more rounds, 5 or more rounds, 10 or more rounds, 15 or more rounds, or for example, 20 or more rounds. By repeating a high-throughput screening, in which 1 or more mutations, 5 or more mutations, or for example, 10 or more mutations are introduced in each round, for example, 10 rounds, mutants with 10 or more mutations, 50 or more mutations, or for example, 100 or more mutations introduced from the starting citrulline oxidase, and which also have activity can be rapidly obtained. Furthermore, by repeating for 20 rounds, mutants with 20 or more mutations, 100 or more mutations, or for example, 200 or more mutations introduced from the starting citrulline oxidase, and which also have activity can be rapidly obtained. Such work can be carried out by repeating a routine process.

[0074] Mutations can be introduced at one or more positions from the first to the last amino acid in the full-length amino acid sequence of citrulline oxidase. However, functionally important regions of the enzyme, such as the active site, substrate recognition site, coenzyme recognition motif, and their vicinity, are excluded. Those skilled in the art are familiar with functionally important regions of the enzyme, including the active site, substrate recognition site, and coenzyme recognition motif of GLOD. Those skilled in the art can also recognize functionally important regions of the enzyme, including the coenzyme recognition motif, based on the teachings herein, for GLOD-based citrulline oxidase. In certain embodiments, for example, one or more mutations can be introduced first at positions 1 to 10 of the full-length sequence of citrulline oxidase. Then, starting from a citrulline oxidase mutant that has been confirmed to have activity, one or more mutations can be further introduced at positions 11 to 20 to confirm activity. This can be repeated n times (n ≤ 65). For example, one or more mutations can be introduced at positions 631 to 640 on the 64th iteration. For example, one mutation can be introduced at position 641 on the 65th iteration. During the process, regions important to the enzyme's function and regions not intended to be modified may be skipped as appropriate. This allows for the introduction of arbitrary mutations at any position in the full-length sequence, excluding regions important to the enzyme's function, and enables the rapid acquisition of active citrulline oxidase mutants having, for example, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or even 200 or more mutations.

[0075] Mutations may be introduced randomly or by rational design. In one embodiment, mutations introduced by rational design or randomly may be conservative amino acid substitutions. Conservative amino acid substitutions include amino acid substitutions in which the amino acid before substitution and the amino acid after substitution have similar chemical properties (e.g., Stryer et al., Biochemistry, 5th edition, 2002, pp. 44-49). For example, conservative amino acid substitutions may be selected from the group consisting of (i) substitution of a basic amino acid with a different type of basic amino acid; (ii) substitution of an acidic amino acid with a different type of acidic amino acid; (iii) substitution of an aromatic amino acid with a different type of aromatic amino acid; (iv) substitution of a nonpolar aliphatic amino acid with a different type of nonpolar aliphatic amino acid; and (v) substitution of a polar uncharged amino acid with a different type of polar uncharged amino acid. Basic amino acids may be selected from, for example, arginine, histidine, and lysine. Acidic amino acids may be, for example, aspartic acid or glutamic acid. Aromatic amino acids may be selected from, for example, phenylalanine, tyrosine, and tryptophan. Nonpolar aliphatic amino acids may be selected from, for example, glycine, alanine, valine, leucine, methionine, and isoleucine. Polar uncharged amino acids may be selected from, for example, serine, threonine, cysteine, proline, asparagine, and glutamine.

[0076] In some embodiments, mutations introduced by rational design or randomly introduced include substitutions of functionally similar amino acids. A table of functionally similar amino acids is widely known in the art. In some embodiments, substitutions of functionally similar amino acids may involve the original amino acid and the substituted amino acid belonging to one of the following amino acid classifications: 1) glycine (G), alanine (A); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K), histidine (H); 5) isoleucine (I), leucine (L), valine (V), proline (P); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0077] In a typical embodiment, conservative amino acid substitutions, or substitutions with functionally similar amino acids, are not present in functionally important regions of citrulline oxidase, such as the active site, substrate recognition site, coenzyme recognition motif, and their vicinity, and therefore do not significantly affect the enzyme's activity.

[0078] Citrulline oxidase mutants may also include those in which additional amino acids are inserted compared to the pre-mutation sequence. In typical embodiments, the amino acid insertions are not located in functionally important regions of the enzyme, such as the active site, substrate recognition site, coenzyme recognition motif, or their vicinity, and therefore do not significantly affect the enzyme's activity. Citrulline oxidase mutants may also include those in which additional amino acids are added compared to the pre-mutation sequence. In some embodiments, the amino acid addition occurs at the N-terminus or C-terminus of citrulline oxidase and does not significantly affect the enzyme's activity. Examples of additions include, but are not limited to, short stretches of histidine residues (e.g., 2-6 histidine residues) to aid in the purification of citrulline oxidase. Other examples of additions include, but are not limited to, the addition of signal peptides to aid in the expression of citrulline oxidase. Examples of signal peptides include known signal sequences or their functional equivalents.

[0079] Citrulline oxidase mutants may also contain amino acid deletions compared to the pre-mutation sequence. In typical embodiments, the amino acid deletions are not located in regions critical to the enzyme's function and therefore do not significantly affect the enzyme's activity. In some embodiments, the deletions may be short, consisting of one or two amino acids. In some embodiments, if the amino acid sequence of one GLOD is compared to the amino acid sequence of another GLOD and an amino acid is deleted in one sequence, that deletion can be introduced into the other GLOD. Since both GLODs exhibit activity, such deletions are unlikely to significantly affect the enzyme's activity. The same applies to citrulline oxidase mutants created based on such GLODs.

[0080] Mutations into citrulline oxidase can be introduced in a way that does not disrupt secondary structures or structural motifs such as α-helix structures or β-sheet structures. The regions of secondary structures can be identified, for example, by secondary structure prediction algorithms. Examples of such algorithms include, but are not limited to, NetSurfP-2.0. The same applies to other structural motifs such as nests and niches.

[0081] The inventors have found that a modified citrulline oxidase, in which amino acid substitutions are introduced at one or more positions corresponding to positions 291, 302, 554, and 221, and positions 502, 479, 305, 310, and 113 of SEQ ID NO: 1, exhibits increased activity toward L-citrulline compared to the original polypeptide. Those skilled in the art will understand, based on these findings, that GLODs from other sources, in which similar amino acid substitutions are introduced at positions corresponding to position 291, etc., of SEQ ID NO: 1, will similarly exhibit increased activity toward L-citrulline and can be used in various reactions.

[0082] In some limited embodiments, reversion mutations from the substituted amino acids in the modified citrulline oxidase to amino acids in the natural GLOD sequence (natural amino acids) are excluded. Furthermore, mutants having a reversion mutation from glutamine to arginine at the amino acid position corresponding to position 291 of SEQ ID NO: 1 are excluded from the modified citrulline oxidase of this disclosure.

[0083] (Corresponding Position) In this specification, when a specific position in a reference amino acid sequence corresponds to a specific position in another similar amino acid sequence, this is referred to as a corresponding position. The amino acid at the corresponding position is also referred to as the corresponding amino acid. For convenience, this specification uses Sequence ID No. 1 as the reference. In this case, the "corresponding position" in the amino acid sequence refers to the position in the amino acid sequence of a polypeptide derived from another species that corresponds to a specific position in the amino acid sequence of Sequence ID No. 1.

[0084] One method for identifying "corresponding positions" in amino acid sequences is to compare amino acid sequences using known algorithms such as the Lippmann-Parson method, thereby maximizing the identity of conserved amino acid residues present in the amino acid sequences of each GLOD or citrulline oxidase. By aligning the amino acid sequences of GLOD or citrulline oxidase in this way, it is possible to determine the position of homologous amino acid residues in each GLOD or citrulline oxidase sequence, regardless of insertions or deletions in the amino acid sequences. Corresponding positions (homologous positions) are thought to exist at the same position in the three-dimensional structure and can be presumed to have similar effects on the specific function of the target GLOD or citrulline oxidase.

[0085] (Corresponding Mutation Locations) In this specification, "the location corresponding to position 291 of the amino acid sequence of SEQ ID NO: 1" refers to the location corresponding to position 291 of SEQ ID NO: 1 when the amino acid sequence of the target GLOD is compared with the amino acid sequence of SEQ ID NO: 1. The same applies to other locations in SEQ ID NO: 1, such as positions 302, 221, and 554, as well as positions 502, 479, 305, 310, and 113.

[0086] (Corresponding Region) The "corresponding region" in the amino acid sequence is defined in the same way as the "corresponding position" described above.

[0087] (Homogeneity, Identity, or Similarity of Amino Acid Sequences) The homology, identity, or similarity of amino acid sequences can be calculated using programs such as Maximum Matching and Search Homology in GENETYX (manufactured by GENETYX Corporation), or Maximum Matching and Multiple Alignment in DNASIS Pro (manufactured by Hitachi Solutions Corporation), or Multiple Alignment in CLUSTAL W. To calculate amino acid sequence identity, when two or more GLODs or citrulline oxidases are aligned, the positions of identical amino acids in those two or more GLODs or citrulline oxidases can be examined. Based on this information, identical regions in the amino acid sequences can be determined. Here, for two or more amino acid sequences, identity % refers to the percentage obtained when the total number of amino acids in the alignable region is used as the denominator and the number of positions occupied by identical amino acids is used as the numerator, when two or more amino acid sequences are aligned using an algorithm such as Blosum62. Therefore, if there is a region in two or more amino acid sequences that shows no identity whatsoever, for example, if one of the amino acid sequences has an additional sequence at the C-terminus that shows no identity whatsoever, then that region of no identity is impossible to align and is therefore not used in calculating the identity percentage.

[0088] Furthermore, the positions of similar amino acids can be investigated in two or more GLODs or citrulline oxidases. For example, multiple amino acid sequences can be aligned using CLUSTALW, in which case Blosum62 is used as the algorithm, and amino acids that are judged to be similar when multiple amino acid sequences are aligned are sometimes called similar amino acids. In the variants of this disclosure, amino acid substitutions may be due to substitutions between such similar amino acids. Through such alignment, it is possible to investigate the regions where the amino acid sequences are identical and the positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, homology regions (conserved regions) in the amino acid sequences can be determined.

[0089] In one embodiment, the modified citrulline oxidase of the present disclosure has, when aligned with GLOD having the amino acid sequence shown in SEQ ID NO: 1, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more of the full-length amino acid sequence identity, the amino acid at the position corresponding to 291 in SEQ ID NO: 1 is substituted with glutamine, and the activity toward L-citrulline is increased compared to the original GLOD.

[0090] In one embodiment, the GLOD variant of the present disclosure has an amino acid sequence in which one or more amino acids are modified or mutated, deleted, substituted, added and / or inserted at positions other than those corresponding to positions 291, 302, 221, 554, and 502, 479, 305, 310, and 113 of SEQ ID NO: 1, the amino acid at the position corresponding to 291 of SEQ ID NO: 1 is substituted with glutamine, and the activity toward L-citrulline is increased compared to the original GLOD. Here, one or more amino acids means 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 4, for example 1 to 3, for example 1 or 2 amino acids.

[0091] Unless otherwise specified, the substituted amino acid introduced at the position corresponding to position 291 of SEQ ID NO: 1 with respect to the modified citrulline oxidase of this disclosure is glutamine. In some embodiments, the substituted amino acid introduced at the position corresponding to position 302 of SEQ ID NO: 1 with respect to the modified citrulline oxidase of this disclosure may be selected from the group consisting of methionine, isoleucine, and leucine. In some embodiments, the substituted amino acid introduced at the position corresponding to position 554 of SEQ ID NO: 1 with respect to the modified citrulline oxidase of this disclosure may be selected from the group consisting of methionine, leucine, asparagine, isoleucine, or glutamine. In some embodiments, the substituted amino acid introduced at the position corresponding to position 221 of SEQ ID NO: 1 with respect to the modified citrulline oxidase of this disclosure may be selected from the group consisting of aspartic acid or glutamic acid. In some embodiments, the substituted amino acid introduced at the position corresponding to position 502 of SEQ ID NO: 1 with respect to the modified citrulline oxidase of this disclosure may be glycine or alanine. In one embodiment, with respect to the modified citrulline oxidase of the present disclosure, the substituted amino acid introduced at the position corresponding to position 479 of SEQ ID NO: 1 may be selected from the group consisting of leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, and threonine. In one embodiment, with respect to the modified citrulline oxidase of the present disclosure, the amino acid substitution introduced at the position corresponding to position 305 of SEQ ID NO: 1 may be selected from the group consisting of valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, and tryptophan. In one embodiment, with respect to the modified citrulline oxidase of the present disclosure, the amino acid substitution introduced at the position corresponding to position 310 of SEQ ID NO: 1 may be selected from the group consisting of valine, leucine, and methionine. In one embodiment, with respect to the modified citrulline oxidase of the present disclosure, the amino acid substitution introduced at the position corresponding to position 113 of SEQ ID NO: 1 may be arginine.

[0092] (Production of Citrulline Oxidase) In one embodiment, the present invention provides a method for producing citrulline oxidase, comprising the steps of culturing a bacterial strain having the ability to produce citrulline oxidase under conditions in which the citrulline oxidase can be expressed, and isolating citrulline oxidase from the culture or culture medium. This method can use host cells transformed with a vector incorporating the gene encoding the citrulline oxidase of the present disclosure. Here, the conditions in which citrulline oxidase can be expressed mean that the citrulline oxidase gene is transcribed and translated, and that the polypeptide encoded by the gene is produced.

[0093] Furthermore, the culture medium used for culturing the above-mentioned bacterial strains may include, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or extract from soybeans or wheat bran, to which one or more inorganic salts such as sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate are added, and carbohydrate raw materials, vitamins, etc. are added as needed.

[0094] The initial pH of the culture medium should be adjusted to pH 7-9. Culturing should be carried out at a culture temperature of 20-42°C, preferably around 25-37°C, for 4-24 hours, and more preferably around 25-37°C, for 8-16 hours, using methods such as deep culture with aeration and stirring, shaking culture, or static culture.

[0095] After culturing is complete, citrulline oxidase can be collected from the culture using conventional enzyme collection methods. For example, the bacterial cells can be subjected to ultrasonic disruption, grinding, or other conventional methods, or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be lysed by shaking or standing in the presence of toluene or the like to release the enzyme from the bacterial cells. Then, the solid portion of this solution can be removed by filtration, centrifugation, etc., and if necessary, nucleic acids can be removed with streptomycin sulfate, protamine sulfate, or manganese sulfate, etc. After that, ammonium sulfate, alcohol, acetone, etc., are added and fractionated, and the precipitate is collected to obtain the crude enzyme.

[0096] To obtain a purified enzyme preparation from the crude enzyme, a purified citrulline oxidase enzyme preparation can be obtained by appropriately selecting or combining methods such as gel filtration using Sephadex, Superdex, or Ultrogel; adsorption elution using ion exchange carriers, hydrophobic carriers, or hydroxyapatite; electrophoresis using polyacrylamide gel; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; or fractionation using molecular sieve membranes or hollow fiber membranes.

[0097] In one embodiment, the modified citrulline oxidase of the present disclosure may, for example, (i) use FAD as a coenzyme, (ii) recognize L-citrulline as a substrate, and (iii) oxidize L-citrulline to produce 2-oxo-5-(carbamoylamino)pentanoic acid, ammonia, and hydrogen peroxide.

[0098] The modified citrulline oxidases described herein shall be excluded if they do not show activity toward L-citrulline.

[0099] (Compositions, Reagents, Electrodes, Sensors, and Kits) In one embodiment, the present invention provides a reagent composition for measuring L-citrulline, a reagent composition for measuring PAD activity, a measuring reagent, an electrode, a sensor, or a kit, all comprising citrulline oxidase. The composition, reagent, electrode, sensor, or kit may contain a reagent for measuring reduced compounds, a reagent for measuring hydrogen peroxide, a buffer, a surfactant, salts, a preservative, etc. Solubilizers, stabilizers, reactivity enhancers, glycated hemoglobin denaturants, reducing agents, bovine serum albumin, sugars (glycerin, lactose, sucrose, etc.), etc. may also be added. The composition, reagent, electrode, sensor, or kit may be further modified as needed to include other known stabilizers, contaminant elimination systems, etc. Technologies used in various conventional reagents, electrodes, sensors, and kits can be appropriately modified and used in the compositions, reagents, electrodes, sensors, or kits of this disclosure.

[0100] Examples of surfactants include nonionic surfactants and ionic surfactants, such as cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0101] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides, and alkyl monoglyceryl ethers.

[0102] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkylbenzyldimethylammonium salts, pyridinium salts (e.g., alkylpyridinium salts), phosphonium salts (e.g., alkylphosphonium salts), imidazolium salts (e.g., alkylimidazolium salts), and isoquinonium salts (e.g., alkylisoquinonium salts).

[0103] The reagent for measuring hydrogen peroxide may contain peroxidase and / or a chromogenic substrate. Examples of chromogenic substrates, in addition to 4-aminoantipyrine, include ADOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine), ALOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline), TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), DA-67 (10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenocyazine), and DA-64 (N-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)-diphenylamine).

[0104] (Method for Measuring L-Citrulline) In one embodiment, the present disclosure provides a method for measuring L-citrulline. The measurement of L-citrulline may be qualitative or quantitative. The quantitative method may include the steps of contacting a sample containing L-citrulline (or a sample that may contain L-citrulline) with the citrulline oxidase of the present disclosure, and measuring the reaction product or consumption. Contact as used in the quantitative method includes all modes of physically bringing the enzyme and the sample together so that the citrulline oxidase can catalyze the oxidation reaction of L-citrulline, and includes not only, for example, mixing a free enzyme with L-citrulline in solution, but also modes of adding or dropping a solution sample containing L-citrulline to an enzyme supported on a solid support.

[0105] The sample used for measurement can be any sample that may contain L-citrulline. The sample may be processed as appropriate. If the sample contains a peptide containing an L-citrulline residue, the sample may be treated with peptidase to release citrulline.

[0106] By keeping the amount of enzyme and reaction time constant and varying the amount of L-citrulline added, the minimum detectable L-citrulline concentration (detection limit) when using the citrulline oxidase can be determined by investigating the citrulline concentration range in which the absorbance of the detected chromogenic substrate decreases proportionally as the amount of L-citrulline decreases. The amount of enzyme and reaction time can be set so that the detection limit is lower than the L-citrulline concentration in the sample or blood, or the concentration of the L-citrulline-containing peptide.

[0107] For quantitative measurements, a calibration curve can be created beforehand by performing regression analysis, such as the least squares method, from measured values ​​such as absorbance of a control containing L-citrulline of known concentration. By plotting the measured values ​​of a sample with an unknown L-citrulline concentration against the created calibration curve, the L-citrulline concentration in the sample can be quantified.

[0108] The time for reacting a sample containing L-citrulline with citrulline oxidase can be, for example, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, less than 60 minutes, less than 30 minutes, less than 10 minutes, for example less than 5 minutes, for example 0.5 minutes or more to less than 60 minutes, 1 minute or more to less than 30 minutes, 1 minute or more to less than 20 minutes, for example 1 minute or more to less than 10 minutes, for example 1 minute or more to less than 5 minutes. The reaction temperature depends on the optimal temperature of the enzyme used, but for example 20 to 45°C, and temperatures used in normal enzymatic reactions can be appropriately selected.

[0109] The amount of citrulline oxidase enzyme used depends on the amount of substrate contained in the sample solution, but for example, it can be added so that the final concentration is 0.1 to 50 U / mL, for example, 0.2 to 10 U / mL. The pH during the reaction can be adjusted using a buffer, taking into consideration the pH at which citrulline oxidase can act, for example, the optimal pH. The reaction pH is, for example, 3 to 11, 5 to 9, for example, 6 to 8.

[0110] The measurement of hydrogen peroxide can be performed simultaneously with the hydrogen peroxide generation process and can proceed concurrently with the action of citrulline oxidase. Instead of the product, a consuming substance may be measured; for example, dissolved oxygen can be measured, and the amount of dissolved oxygen in the reaction solution can be measured using a dissolved oxygen meter or the like.

[0111] (Method for measuring citrulline oxidase activity) The following is an example of a method for measuring citrulline oxidase activity using L-citrulline as a substrate, but the method is not limited to this. L-citrulline may be commercially available. In this specification, unless otherwise specified, enzyme titer is defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute when measured with L-citrulline as a substrate at 30°C and pH 7.4, with 1 U being the amount of enzyme.

[0112] A: Reagent for activity measurement (Reagent 1) 250 mM potassium phosphate buffer, pH 7.4 (Reagent 2) 30 mM 4-aminoantipyrine (4-AA) solution (Reagent 3) 15 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) solution (Reagent 4) 300 U / mL horseradish peroxidase (POD) solution (Reagent 5) 300 mM L-citrulline solution B: Activity measurement method Mix 300 µL of Reagent 1, 12.5 µL of Reagent 2, 25 µL of Reagent 3, 12.5 µL of Reagent 4, (375-V) µL of deionized water, and V µL of L-citrulline solution, and incubate the mixture at 30°C for 5 minutes. Then, add 25 µL of Reagent 5 and mix well, then measure the absorbance (A ５５５ ) of light at a wavelength of 555 nm using a spectrophotometer U-3900 (manufactured by Hitachi High-Tech Science) with a cell holder incubated at 30°C, and calculate the change in A ５５５ per minute (ΔA Ｓ ). As a control experiment, 25 µL of deionized water is added instead of 25 µL of Reagent 5, the absorbance (A ５５５ ) of light at a wavelength of 555 nm is measured, and the change in A ５５５ per minute (ΔA ０ ) is calculated.

[0113] Oxidase activity (U / mL) can be calculated based on the following formula. "39.2" in the formula represents the millimolar extinction coefficient (mM －１ cm －１ ) of the quinoneimine dye formed by condensation of 4-AA and TOOS for light at a wavelength of 555 nm. [Formula] U / mL = (ΔA Ｓ - ΔA ０ ) × 600 × df / (39.2 × 0.5 × V) = 30.6 × (ΔA Ｓ - ΔA ０ ) × df / V When different coloring reagents are used, a wavelength corresponding to the coloring reagent and the millimolar extinction coefficient at that wavelength can be used.

[0114] (Evaluation of substrate specificity of citrulline oxidase) The substrate specificity of citrulline oxidase can be evaluated using the following values: "Glu / Cit," which is calculated by dividing the activity measured with L-glutamic acid as a substrate by the activity measured with L-citrulline as a substrate; "His / Cit," which is calculated by dividing the activity measured with L-histidine as a substrate by the activity measured with L-citrulline as a substrate; "Leu / Cit," which is calculated by dividing the activity measured with L-leucine as a substrate by the activity measured with L-citrulline as a substrate; and / or "Phe / Cit," which is calculated by dividing the activity measured with L-phenylalanine as a substrate by the activity measured with L-citrulline as a substrate. If the values ​​of "Glu / Cit," "His / Cit," "Leu / Cit," and / or "Phe / Cit" decrease after introducing amino acid substitutions to citrulline oxidase compared to before the substitution, it indicates that the substrate specificity of citrulline oxidase has improved. The substrate concentration used for activity measurement may be 0.5 to 10 mM, for example, 10 mM or 1 mM.

[0115] If the Glu / Cit ratio of the original citrulline oxidase is 100, and the Glu / Cit ratio of the modified citrulline oxidase is 50 under the same measurement conditions, then the substrate specificity of the modified citrulline oxidase is considered to have decreased to 50% compared to the original citrulline oxidase (100%). The same applies to other substrate specificities. In one embodiment, citrulline oxidase modified by introducing amino acid substitutions may have a Glu / Cit ratio of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, or, for example, 0.01% or less, compared to the original citrulline oxidase (100%). In one embodiment, citrulline oxidase modified by introducing amino acid substitutions can be modified to have a His / Cit ratio of 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3.5% or less, 3% or less, 2.7% or less, 2.6% or less, 2.5% or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.13% or less, 0.1% or less, 0.06% or less, 0.05% or less, for example, 0.01% or less, compared to the original citrulline oxidase (100%). In one embodiment, citrulline oxidase modified by the introduction of amino acid substitutions may have its Phe / Cit ratio modified to 50% or less, 45% or less, 41% or less, 40% or less, 30% or less, 25% or less, 24% or less, 22% or less, 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, for example, 0.01% or less, compared to citrulline oxidase before modification (100%). In another embodiment, citrulline oxidase modified by the introduction of amino acid substitutions may have its Leu / Cit ratio modified to 50% or less, 40% or less, 32% or less, 30% or less, 25% or less, 24% or less, 23% or less, 20% or less, 16% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, for example, 0.01% or less, compared to citrulline oxidase before modification (100%).In one embodiment, citrulline oxidase modified by introducing amino acid substitutions can have its Leu / Cit ratio modified to 50% or less, 40% or less, 30% or less, 25% or less, 24% or less, 21% or less, 20% or less, 18% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.1% or less, for example, 0.01% or less, compared to the original citrulline oxidase (100%). In one embodiment, citrulline oxidase modified by the introduction of amino acid substitutions may have its Phe / Cit ratio modified to 50% or less, 40% or less, 30% or less, 25% or less, 22% or less, 20% or less, 15% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7.9% or less, 7% or less, 6.6% or less, 6.5% or less, 6.2% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.1% or less, for example, 0.01% or less, compared to the original citrulline oxidase (100%). In case of any doubt, the substrate specificity such as Glu / Cit as used herein refers to the substrate specificity based on the value obtained when activity is measured at a substrate concentration of 1 mM.

[0116] In one embodiment, the Disclosure provides a method for quantifying citrulline using a modified citrulline oxidase. The modified citrulline oxidase may be one of those described herein. For example, the modified citrulline oxidase of the Disclosure may be contacted with a sample containing L-citrulline or a sample that may contain L-citrulline, and the resulting hydrogen peroxide may be measured. The hydrogen peroxide may be measured by known methods or by the methods described herein. In one embodiment, the method of the Disclosure does not involve medical procedures. In one embodiment, the method of the Disclosure does not involve human diagnostic methods.

[0117] (Verification of citrullination of substrate peptides used in the PAD reaction) Recombinant protein arginine deiminase 4 (Funakoshi Co., Ltd.) was used for the PAD reaction. The substrate peptides were synthesized by a contract manufacturer (GenScript Japan Co., Ltd.). The reaction was carried out by mixing 100 ppm of the substrate peptide with 100 mM HEPES buffer pH 7.5, 500 mM CaCl2, and a 10-fold diluted PAD solution. The peptide sequences examined are shown in Table 1 (SEQ ID NOs. 14-36). Further peptides examined are also shown in Table 1 (SEQ ID NOs. 62-79). All amino acids constituting the peptides are L-forms. KN-301 was obtained from DOJINDO. For the structure of KN-301, please refer to Biological Data Analysis vol. 4, No. 2 (2024).

[0118]

[0119] The reaction solution was incubated at 37°C for 10–60 minutes, then diluted 100-fold (to a final substrate peptide concentration of 1 ppm), and filtered. A DISMIC 13CP020AN filter (Advantec) was used. The filtered solution was stored at 4°C in a screw vial (with a 0.3 mL glass insert, Thermo Fisher Scientific). Mass spectrometry was performed on these samples. For mass spectrometry, (i) LC-MS / MS (LCMS-8050, Shimadzu Corporation) and (ii) LC-TOFMS (Xevo G2-XS QTOF, Waters Corporation) were used. The measurement conditions for each are shown in Tables 2 and 3.

[0120]

[0121]

[0122] The concentration of the substrate peptide was calculated from the peak area of ​​the resulting chromatogram using a calibration curve prepared with peptide solutions of known concentrations. The citrullination rate was evaluated using two methods: (i) the consumption rate of the L-arginine-containing peptide and (ii) the production rate of the L-citrulline-containing peptide. Specifically for (i), the rate was calculated as follows: [Equation 1] Substrate peptide consumption rate (%) = 1 - {(peak area of ​​the substrate peptide after PAD addition) / (*peak area of ​​the control substrate peptide)}. The control refers to the peak area when only buffer was added and incubated in the same way as the sample. For (ii), the rate was calculated as follows: Substrate peptide consumption rate (%) = {(peak area of ​​the substrate peptide after PAD addition) / (*peak area of ​​the control substrate peptide)}.

[0123] (Test Results: Verification of Citrullination of Substrate Peptides Used in PAD Reaction) The PAD reaction was performed using 15 types of substrate peptides (No. 1 to 15). Recombinant protein arginine deiminase 4 (Funakoshi Co., Ltd.) was used as PAD4. Table 4 shows the citrullination rate (consumption rate of L-arginine-containing peptide) after 60 minutes of reaction following the addition of PAD4.

[0124]

[0125] The citrullination rate varied significantly depending on the sequence of the substrate peptide, with No. 9: MDRDG peptide showing the highest calculated citrullination rate. Subsequently, Table 5 shows the results of comparing the citrullination rates of a total of eight peptides, including five peptides (Nos. 16-20) and three peptides (Nos. 3, 9, and 13) that showed high citrullination rates at 60 minutes, using reaction times shorter than 60 minutes.

[0126]

[0127] In both reaction times of 10 minutes and 30 minutes, the MDRDG peptide showed the highest citrullination rate. These results suggest that the MDRDG peptide is suitable as a substrate peptide for PAD4. Subsequently, a calibration curve was created using No. 21: MD{Cit}DG peptide, which is the PAD reaction product of No. 9: MDRDG peptide, and the production rate of L-citrulline-containing peptides was also verified. The results are shown in Table 6.

[0128]

[0129] These results confirmed that the tendency for L-arginine-containing peptides to be consumed coincided with the tendency for L-citrulline-containing peptides to be produced. Based on these results, it was determined that using the consumption rate of L-arginine-containing peptides as an indicator for substrate peptide selection is appropriate.

[0130] (Test Results: Optimal Sequence Verification of MDRDG as a Substrate Peptide) Next, No. 22: SDRDG and No. 23: GDRDG, which were created by substituting the leading amino acid of No. 9: MDRDG peptide, were synthesized under contract, and the optimality of the substrate peptide sequence was examined in more detail. Table 7-1 shows the comparison results of the three peptides.

[0131]

[0132] These results suggest that MDRDG exhibited the best reactivity, and that the initial amino acid sequence is also important for the PAD reaction. Through a series of studies, it was determined that the MDRDG peptide is the optimal substrate peptide for PAD4; therefore, the following examples will focus on the MDRDG peptide. Furthermore, by adjusting the reaction time, the citrullination reaction proceeded sufficiently with peptides other than MDRDG, demonstrating that other peptides can also be used. Specifically, it was shown that the first amino acid residue X1 of the pentapeptide (X1-X2-Arg-X4-X5) can be Met or Ser. It was also shown that the second amino acid residue X2 of the pentapeptide can be Asp, Glu, Gly, or Ser. Additionally, it was shown that the fourth amino acid residue X4 of the pentapeptide can be Asp, Gly, or Met. Finally, it was shown that Gly is preferable for the fifth amino acid residue X5 of the pentapeptide.

[0133] (Searching for peptide sequences for PAD2 detection) Next, a similar verification was performed for PAD2, a different type of PAD. Figure 7 shows the absorbance changes of each of the three peptides when substrate peptides for PAD4, No. 4: MSRMG, No. 9: MDRDG, or No. 19: MERMG, were added. When the PAD2 concentration was 35 μg / mL, peptide No. 4 showed a higher value compared to peptides No. 19 and No. 9. This trend was also observed when the PAD2 concentration was 3.5 ug / mL (Figure 8). From these results, it was suggested that peptide No. 4: MSRMG is a peptide sequence that shows high reactivity to PAD2.

[0134] To find substrate peptides more specific to PAD2, the amino acid residue at the first amino acid residue X1 of the 5-residue peptide No. 4: MSRMG was modified. When the consumption of L-arginine-containing peptides was compared after changing the amino acid at the first amino acid residue X1, peptides No. 26: HSRMG and No. 27: KSRMG showed reaction rates equivalent to peptide No. 4 (Table 7-2). From these results, peptides No. 26 and No. 27 were identified as highly reactive substrate peptides for PAD2.

[0135]

[0136] Next, the reactivity of three peptides, No. 28, 29, and 30, in relation to PAD4 / PAD2 was compared to search for a substrate peptide that reacts more specifically with PAD2. The results of the comparison are shown in Figure 9. The PAD4 / PAD2 ratio (%) was 144% for peptide No. 28, 96% for peptide No. 30, and reduced to 21% for peptide No. 29. From these results, peptide No. 29: KS{Cit}MG showed higher reactivity to PAD2. From these results, it is suggested that the amino acid of the first amino acid residue X1 of the substrate peptide is preferably Lys.

[0137] Next, the amino acid at the fourth amino acid residue X4 of the substrate peptide was changed. Due to the physical properties of the substrate peptide, the PAD activity of each peptide substrate was evaluated by comparing the absorbance change after the addition of the second reagent in the PAD measurement test (three-step test) system, which will be described later in the section on seamless integration of the three-step measurement system below, while varying the type and concentration of the PAD sample and the type of substrate peptide. When peptide No. 27 was subjected to this evaluation system, a new problem arose: the signal increased in the absence of PAD. Therefore, four peptides were prepared by changing the amino acid at the fourth amino acid residue X4 of peptide No. 27, with the aim of preventing an increase in background signal in the absence of PAD, and these were compared. The results are shown in Figure 10. From these results, it was shown that the background signal was suppressed when the amino acid at the fourth amino acid residue X4 of the peptide was I, V, or K. Furthermore, peptide No. 32 showed high reactivity, with the reaction starting around 10 minutes into the reaction. From these results, it was shown that peptide No. 32 is a more appropriate substrate peptide. It was also shown to have reactivity to PAD2.

[0138] Figure 11 shows the results of comparing PAD2 specificity. Peptide No. 32 was reactive not only to PAD2 but also to PAD4, and although the background signal decreased and the reactivity improved, it was suggested that the specificity for PAD2 observed with peptide No. 27 had decreased. Therefore, we decided to change the amino acid of the second amino acid residue X2 of the substrate peptide.

[0139] The specificity of four peptides, each with a modified amino acid at the second amino acid residue X2 of the substrate peptide, was compared using a three-step test. The results are shown in Figure 12. These results indicate that specificity for PAD2 is observed when the amino acid at the second amino acid residue X2 is W, I, Q, etc. Peptide No. 35 (KWRKG) showed very good reactivity and good specificity. Peptide No. 36 (KIRKG) showed moderate reactivity and very good specificity. Peptide No. 37 (KPRKG) showed almost no reactivity (comparative example). Peptide No. 38 (KQRKG) showed very high specificity and very high reactivity, indicating that the amino acid at the second amino acid residue X2 of the peptide being Gln is important for both PAD2 specificity and reactivity. Peptide No. 35 (KWRKG) was able to detect PAD2 down to 1 μg / mL, although it reacted similarly to PAD4 at 4 μg / mL. Peptide No. 38 (KQRKG) was capable of detecting PAD2 down to 1 μg / mL and also showed low reactivity to PAD4, indicating high specificity for PAD2 (Figure 13).

[0140] (Search for peptidases that degrade L-citrulline-containing peptides after the PAD reaction) We investigated peptidases that degrade L-citrulline-containing peptides produced by the PAD reaction and release L-citrulline. The 11 peptidases listed at the beginning of Table 8 were used for the investigation. In addition, carboxypeptidase A and dispase listed at the end of Table 8 were also investigated.

[0141]

[0142] (Comparative Verification of Peptidase Activity of Citrullinated Peptides) MD{Cit}DG was prepared by replacing L-arginine with L-citrulline in the MDRDG peptide, which was selected as a substrate peptide suitable for the PAD reaction. 1000 ppm of MD{Cit}DG peptide was mixed with 100 mM HEPES buffer pH 7.5, 200 μM CoCl2, and various peptidases, and incubated at 37°C for 10 minutes. After incubation, 6 mg / mL 4-aminoantipyrine (hereinafter 4-AA, manufactured by Fujifilm Wako Pure Chemical Industries), 15 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (hereinafter TOOS, manufactured by Dojin Chemical Laboratories), 300 U / mL peroxidase (hereinafter POD, manufactured by Toyobo), and the citrulline oxidase (CitOX) developed in this study were added to the incubated samples. The absorbance at 555 nm was measured every 15 minutes using a plate reader Infinite M Plex (TECAN), and the progress of each peptidase reaction was compared. The results of comparing the activity of 11 peptidases using absorbance at 555 nm are shown in Table 9-1.

[0143]

[0144] (Verification of enhanced peptidase activity when dispase is added) GD{Cit}DG was prepared by replacing L-arginine with L-citrulline in the GDRDG peptide, which was selected as a substrate peptide suitable for the PAD reaction. 1000 ppm of MD{Cit}DG peptide was mixed with 100 mM HEPES buffer pH 7.5, various peptidases, 0.1 mM KN-301 (DOJINDO), 300 U / mL peroxidase (hereinafter POD, manufactured by Toyobo), and the citrulline oxidase (CitOX) developed in this study. After 30 minutes, the absorbance at 665 nm was measured using a plate reader Infinite M Plex (TECAN), and the progress of the various peptidase reactions was compared. The evaluation results are shown in Table 9-2.

[0145]

[0146] Since all the peptidases tested showed absorbance, it was confirmed that all of them can be used to release L-citrulline from peptides. In particular, 4: Pfu aminopeptidase I showed the highest activity, followed by 6: porcine kidney microsome-derived leucine aminopeptidase and 7: recombinant human methionine aminopeptidase 1 / METAP1 protein, which also showed high activity. Furthermore, it was found that when dispase, an endopeptidase, was added together with exopeptidases such as leucine aminopeptidase, the increase in absorbance increased, promoting peptide degradation. See Figure 14. The increase in absorbance was significantly larger for all of carboxypeptidase A (CPA), carboxypeptidase B (CPB), and leucine aminopeptidase (LAP) when dispase was added. Based on these results, Pfu-aminopeptidase I was determined to be the optimal peptidase for degrading peptides after the PAD reaction, and therefore, we will focus on it in subsequent studies. Furthermore, these results suggest that equivalents, similar substances, or homogeneous compounds of the above peptidase can also be used in a similar manner.

[0147] (Creation of an enzyme for detecting L-citrulline) The enzyme for detecting L-citrulline (citrulline oxidase) was created by introducing site-directed mutations into the glutamate oxidase gene. Using "pET22b-M7GLODΔ49C-T7E," the expression plasmid for the glutamate oxidase "M7GLODΔ49C-T7 / R186E" disclosed in International Publication No. 2024 / 090562, as a template, a modified M7GLODΔ49C-T7E expression plasmid was obtained by introducing site-directed mutations. In this specification, M7GLODΔ49C-T7 / R186E will be referred to as M7GLODΔ49C-T7E.

[0148] For site-directed mutagenesis, the PCR reaction mixture was prepared by mixing 10 μL of KOD one PCR Master Mix (Toyobo), 3 μL of 2 μM Fw primer, 3 μL of 2 μM Rv primer, 0.5 μL of 40 μg / mL template DNA, and 3.5 μL of deionized water. The PCR reaction conditions were 15 cycles of "98°C for 10 seconds → 55°C for 5 seconds → 68°C for 35 seconds". After PCR, 1 μL of DpnI was added to the reaction mixture and incubated at 37°C for 30 minutes. The E. coli JM109 strain was transformed using this solution. The plasmid sequences extracted from the resulting transformants were cultured and their sequences were confirmed to be as intended by DNA sequencing analysis. The names of the created mutants and the combinations of Fw and Rv primers used in PCR are shown in Table 10.

[0149]

[0150] (Recombinant production of enzyme) A production strain of M7GLODΔ49C-T7E or a modified M7GLODΔ49C-T7E was inoculated into 2.5 mL of LB-amp medium (ampicillin concentration 50 μg / mL) in a test tube and cultured overnight at 37°C and 180 rpm. 2.5 mL of the seed culture solution was inoculated into 250 mL of LB-amp medium (ampicillin concentration 100 μg / mL) in a Sakaguchi flask and cultured at 37°C and 130 rpm. When the OD600 of the culture solution reached 0.6 to 1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM or 0.1 mM, and cultured at 15°C and 130 rpm for 16 hours.

[0151] The culture medium was centrifuged at 8,000 rpm for 10 minutes to obtain a pellet, which was then resuspended in 4–25 mL of 10 mM potassium phosphate buffer (PPB) at pH 6.0. After sonication of the bacterial suspension, the supernatant obtained by centrifuging at 15,000 rpm for 15 minutes was collected and used as the crude enzyme solution for M7GLODΔ49C-T7E or modified M7GLODΔ49C-T7E.

[0152] (Enzyme Purification) Modified M7GLODΔ49C-T7E crude enzyme solution was mixed with 250 mM potassium phosphate buffer (PPB) pH 6.0, and the buffer concentration was adjusted to 100 mM. The mixture was then incubated in a 60°C water bath for 30 minutes. The supernatant obtained by centrifugation at 10,000 rpm for 15 minutes was collected, and ammonium sulfate was added to a final concentration of 0.7 M. The enzyme solution was filtered through a 0.22 μm filter, and the filtrate was subjected to hydrophobic interaction chromatography.

[0153] A HiScreen-Butyl-FF column (Cytiva) was used for hydrophobic interaction chromatography. The enzyme solution was loaded onto the column, which had been equilibrated with 20 mM potassium phosphate buffer (PPB) pH 6.0 containing 0.7 M ammonium sulfate. After washing with the buffer used for equilibration, gradient elution was performed while decreasing the ammonium sulfate concentration to 0 M. The purity of each fraction was evaluated by SDS-PAGE, and high-purity fractions were recovered.

[0154] The recovered fraction solution was dialyzed to 20 mM MES buffer pH 6.0, and then the enzyme solution was concentrated to the appropriate concentration by ultrafiltration.

[0155] (Measurement of Oxidase Activity) 4-aminoantipyrine (4-AA) (manufactured by Fujifilm Wako Pure Chemical Industries), TOOS (manufactured by Dojin Chemical Laboratories), and horseradish peroxidase (POD) (manufactured by Toyobo) were used as reagents for measuring oxidase activity. The reagent compositions for activity measurement are shown in Table 11. For dilution of the enzyme solution (e.g., GLOD solution or citrulline oxidase solution), 10 mM PPB (pH 7.5) containing 0.15% bovine serum albumin (BSA, manufactured by Sigma-Aldrich) was used.

[0156]

[0157] After incubating 725 μL of the reagents listed in Table 11 at 30°C for 5 minutes, the substrate amino acid solution was added and mixed to a final concentration of 10 mM. The absorbance of light at a wavelength of 555 nm was measured using a spectrophotometer U-3900 (Hitachi High-Tech Science) with the cell holder incubated at 30°C. ５５５) Measure and A per minute ５５５ Change (ΔA) Ｓ ) was calculated. Measurements were also performed with deionized water added instead of the substrate solution, and the A per minute was calculated. ５５５ Change (ΔA) ０ ) was calculated.

[0158] (Test results: Improvement in specificity, affinity, and activity for L-citrulline) For each mutant, the relative activity to other substrates (for example, relative activity to L-glutamate was expressed as Glu / Cit) was measured with the activity to L-citrulline set to 1. In the mutant with the R291Q substitution, the activity to L-glutamate, the substrate of the template glutamate oxidase, was almost completely eliminated, while activity to L-citrulline was produced. The R291Q mutant had a relative activity to L-histidine (His / Cit) of about 15.3, but since the activity to L-glutamate, the substrate of the template glutamate oxidase, was almost completely eliminated, it was considered to be sufficiently usable for L-citrulline detection. In particular, when the substrate peptide does not contain L-histidine residues, the relative activity to L-histidine is not a problem, so it was considered to be sufficiently usable for L-citrulline detection. Furthermore, when mutants with an amino acid substitution at the G302 position in addition to the R291Q substitution were evaluated, the His / Cit ratio was reduced to as low as 0.414 in the lowest case, improving specificity for L-citrulline.

[0159] The obtained R291Q / G302M mutant showed an affinity Km of 303 mM for L-citrulline, and also exhibited relative activity of approximately 0.4 for L-phenylalanine in addition to L-histidine. Since the activity for L-glutamate, the substrate of the glutamate oxidase used as a template, was almost completely lost, the R291Q / G302M mutant was considered to be sufficiently usable for L-citrulline detection. Furthermore, when an amino acid substitution was introduced at the N221 position in the R291Q / G302M mutant, the Km for L-citrulline improved to 227 mM. When a mutant with an additional amino acid substitution at the E554 position was evaluated, the mutants in which E554 was substituted with methionine and asparagine showed a dramatic decrease in relative activity for L-histidine, an improvement in specificity with relative activity for L-phenylalanine decreasing to approximately 0.15, and a significant improvement in Km for L-citrulline to around 10 mM. Furthermore, the mutant in which E554 was replaced with leucine showed increased relative activity toward L-phenylalanine, and although substrate specificity was slightly reduced, the Km value was significantly improved to 3.51 mM.

[0160] Further improvements in Km were observed in E554M / N221D / G302M / R291Q mutants, specifically in those where S502 was replaced with glycine, and in those where F479 was replaced with leucine, alanine, serine, tryptophan, or methionine. Km was also improved in E554M / N221D / G302M / R291Q mutants where S502 was replaced with alanine. Additionally, Km was improved in mutants where F479 was replaced with glutamine, valine, or threonine.

[0161] Furthermore, Km was improved in the S502G / E554M / N221D / G302M / R291Q mutants in which D305 was replaced with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, in which A310 was replaced with valine or methionine, and in which S113 was replaced with arginine. In addition, Km was improved in the S502G / E554M / N221D / G302M / R291Q mutant in which A310 was replaced with leucine.

[0162]

[0163]

[0164] (Study on seamless integration of the three steps of the measurement system) Using MDRDG, selected as the substrate peptide for PAD4, Pfu aminopeptidase I, selected as the peptidase, and a mutant identified as a useful citrulline oxidase, we constructed a system that seamlessly links three steps: (i) citrullination of the peptide, (ii) release of L-citrulline by the peptidase, and (iii) detection of the released L-citrulline. We then investigated whether PAD4 in the sample could be detected.

[0165] Figure 1 shows the procedure and materials used in this test. Detection was performed by a colorimetric assay using peroxidase / hydrogen peroxide (absorbance at 555 nm was detected using a microplate reader).

[0166] The composition and order of addition of the reagents are shown in Tables 13 and 14. The solution containing PAD4 was used as the sample solution. The solution obtained by adding citrulline oxidase to the reaction solution in step (i) was designated as the first reagent (Table 13), and the peptidase reaction solution from step (ii) and the peroxidase reaction solution used for detection in step (iii) were designated as the second reagent (Table 14).

[0167]

[0168]

[0169] After adding the first reagent, the mixture was incubated at 37°C for 30 minutes. Then, the second reagent was added, and the absorbance at 555 nm was measured every 15 minutes while incubating at 37°C. The measurements were performed using the Infinite M Plex fluorescence multimode plate reader (TECAN), which is available in the cell laboratory.

[0170] (Test Results: Study on Seamless Integration of the Three-Step Measurement System) Figure 2 shows the results of comparing the absorbance change at 555 nm in a PAD sample and a control sample without PAD. The E554M mutant was used as citrulline oxidase. When commercially available PAD4 was added to the PAD4 sample at a final concentration of 4.4 μg / mL, a difference in absorbance began to appear 15 minutes after the addition of the second reagent compared to the control without PAD4. Furthermore, when the concentration of added PAD4 was changed, concentration-dependent absorbance was confirmed 30 minutes after the addition of the second reagent (Figure 3). Similarly, when the reaction was carried out using peptide No. 38 (KQRKG) with PAD2 concentrations of 1, 2, or 4 μg / mL, absorbance increased in accordance with the addition of PAD2, and concentration-dependent absorbance was confirmed 60 minutes after the addition of the second reagent (R 2 (=0.9997). These results demonstrate that PAD on a μg / mL scale can be detected using this system.

[0171] Next, we investigated whether PAD detection was possible with other citrulline oxidase mutants. Figures 4-6 show the changes in absorbance when the E554L mutant and E554N mutant were used in place of the E554M mutant in the same test. In all cases, the condition with PAD added showed higher absorbance compared to the condition without PAD. These results indicate that any of the citrulline oxidases can be used for PAD4 detection on a μg / mL scale. Furthermore, it was shown that PAD4 at concentrations below 1 μg / mL can be detected if the sample volume is sufficient, or by adjusting the reagent concentration and allowing sufficient reaction time.

[0172] PAD activity can be measured using the methods, peptides, and / or citrulline oxidase described herein. This can be used to diagnose rheumatoid arthritis, Alzheimer's disease, cancer, ulcerative colitis, inflammatory bowel disease, fibrosis, and / or multiple sclerosis.

[0173] This specification references numerous documents, including patent applications and manufacturers' manuals. While the disclosures of these documents are not considered relevant to the patentability of the present invention, their entirety is incorporated herein by reference. More specifically, all referenced documents are incorporated herein by reference in the same manner as each individual document is specifically and individually indicated as being incorporated by reference.

[0174] Brief explanation of sequence listings: Sequence ID 1: Amino acid sequence of M7GLODΔ49C-T7E Sequence ID 2: Base sequence of M7GLODΔ49C-T7E Sequence IDs 3-13: Primer sequences Sequence IDs 14-36: Peptide sequences Sequence ID 37: Amino acid sequence of M7GLODΔ49C-T7E / E554N / N221D / G302M / R291Q Sequence IDs 38-61: Primer sequences Sequence IDs 62-76: Peptide sequences

[0175] [Sequence Listing] Amino acid sequence of Sequence ID 1 M7GLODΔ49C-T7E MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFYNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNETQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVNQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSRLHLAFMHSFLGRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGEAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0176]

[0177] Sequence ID 3: Primer sequence aatatgacaa gtcagttgca tttggcattt Sequence ID 4: Primer sequence acttgtcata ttctcgatgg Sequence ID 5: Primer sequence cacagcttct taatgcgcag tgacattgac Sequence ID 6: Primer sequence cacagcttct taattcgcag tgacattgac Sequence ID 7: Primer sequence taagaagctg tgcataaatg c Sequence ID 8: Primer sequence tctgatatgg tggaccaagc tttagaaccg Sequence ID 9: Primer sequence caccatatca gaaactgtca aac Sequence ID 10: Primer sequence tacgcctgcg gtatggccgc agtttacaca Sequence ID 11: Primer sequence tacgcctgcg gtctggccgc agtttacaca Sequence ID 12: Primer sequence tacgcctgcg gtaacgccgc agtttacaca Sequence ID 13 Primer sequence accgcaggcg taggggtc

[0178] Sequence ID 14 SERMG Sequence ID 15 SGRDG Sequence ID 16 SSRGG Sequence ID 17 MSRMG Sequence ID 18 MDRMG Sequence ID 19 SDRMG Sequence ID 20 MGRMG Sequence ID 21 MSRDG Sequence ID 22 MDRDG Sequence ID 23 MGRDG Sequence ID 24 MSRGG Sequence ID 25 SERDG Sequence ID 26 MDRGG Sequence ID 27 MGRGG Sequence ID 28 MERGG Sequence ID 29 SSRMG Sequence ID 30 SGRMG Sequence ID 31 SSRDG Sequence ID 32 MERMG Sequence ID 33 MERDG Sequence ID 34 MD{Cit}DG Sequence ID 35 SDRDG Sequence ID 36 GDRDG

[0179] The amino acid sequence of Sequence ID No. 37, M7GLODΔ49C-T7E / E554N / N221D / G302M / R291Q, is MDDKTYQQLARELLLVGPEPANEDLKLRYLDVLIDNGLEPPVDRKRILIVGAGIAGLVAGHLLTRAGHDVTILEANANRVGGRIKTYHAKKGEPAPFTDPAQIAEAGAMRLPSFHPLTLALIDKLGLKRRLFYNVDIDPKTGNQGAALPPVVYKSFKDGKTWTYGKPSPEFREPDKRNHTWIRTNETQVRRAQYVKDPSAINEGFHLTGCESRLTVSDMVDQALEPVRDYYSVLQSDGRRVNKPFKEWLDGWAGVIRDFDGFSMGRFLREYAGFSDEAVEAIGTIENMTSQLHLAFMHSFLMRSDIDPSATYWEIEGGSR QLPEALAKDLRDQIVMGQRMVRLEYYDPGRDGHHGGLAGPSGPAVAIETVPENEPSAEPQTWTADLAIVTVPLSSLRFVAVTPPFSYKKRRAVIETHYDQATKVLLEYSRRWWEFTEEDWKRELDAIAPGLYEYYQDAAEPPATQVHGGGSTTDNPNRFM YYPSHAVPGSKGGVVLAAYSWSDDAARWDSFDDAERFGYALENLQSVHGRRIEVFYTGAGQTQSWLRDPYACGNAAVYTPHQMTSFHLDVVRPEGPVYFAGEHVSLKHAWIEGAVETAVRAAIAVNEAPVPYDTAAARAEAPRERAGTASATRTREKAVTS

[0180] Sequence ID 38 GCGTATAGTTGGggtGACGATGCAGCGCGT Sequence ID 39 CCAACTATACGCGGCCAAAAC Sequence ID 40 AATCCAAACCGTatgATGTATTATCCGAGT Sequence ID 41 AATCCAAACCGTctgATGTATTATCCGAGT Sequence ID 42 AATCCAAACCGTgcgATGTATTATCCGAGT Sequence ID 43 AATCCAAACCGTtcgATGTATTATCCGAGT Sequence ID 44 AATCCAAACCGTtggATGTATTATCCGAGT Sequence ID 45 ACGGTTTGGATTGTCAGTTGTTG Sequence ID 46 TTAatgCGCAGTgttATTGACCCCAGCGCG Sequence ID 47 TTAatgCGCAGTaatATTGACCCCAGCGCG Sequence ID 48 TTAatgCGCAGTatcATTGACCCCAGCGCG Sequence ID 49 TTAatgCGCAGTctgATTGACCCCAGCGCG Sequence ID 50 TTAatgCGCAGTcaaATTGACCCCAGCGCG Sequence ID 51 TTAatgCGCAGTatgATTGACCCCAGCGCG Sequence ID 52 TTAatgCGCAGTcacATTGACCCCAGCGCG Sequence ID 53 TTAatgCGCAGTaccATTGACCCCAGCGCG Sequence ID 54 TTAatgCGCAGTttcATTGACCCCAGCGCG Sequence ID 55 TTAatgCGCAGTtggATTGACCCCAGCGCG Sequence ID 56 ACTGCGcatTAAGAAGCTGTGC Sequence ID 57 ATTGACCCCAGCgtgACATATTGGGAGATT Sequence ID 58 ATTGACCCCAGCatgACATATTGGGAGATT Sequence ID 59 GCTGGGGTCAATGTCACTGCGc Sequence ID 60 ATGCGTTTGCCTcgtTTCCATCCCTTAACGTTAGCG Sequence ID 61 AGGCAAACGCATAGCTCC

[0181] Sequence ID 62 DSRMG, Sequence ID 63 ESRMG, Sequence ID 64 HSRMG, Sequence ID 65 KSRMG, Sequence ID 66 MS{Cit}MG, Sequence ID 67 KS{Cit}MG, Sequence ID 68 HS{Cit}MG, Sequence ID 69 KSRIG, Sequence ID 70 KSRKG, Sequence ID 71 KSRLG, Sequence ID 72 KSRVG, Sequence ID 73 KWRKG, Sequence ID 74 KIRKG, Sequence ID 75 KPRKG, Sequence ID 76 KQRKG

Claims

1. A method for measuring peptidylarginine deiminase (PAD) activity in a sample, comprising: (i) contacting a sample with a substrate peptide containing an L-arginine residue to cause peptidylarginine deiminase (PAD) contained in the sample to act on the substrate peptide containing the L-arginine residue, thereby generating a peptide containing an L-citrulline residue; (ii) digesting the peptide containing the L-citrulline residue generated in step (i) using a peptidase to release L-citrulline; and (iii) oxidizing the L-citrulline released in step (ii) using a citrulline oxidase and measuring the activity.

2. The method according to claim 1, wherein PAD is PAD4.

3. The method according to claim 2 for diagnosing rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, or for assisting in the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease, wherein the sample is a biological sample obtained from a subject, the method comprising measuring the PAD4 activity of the biological sample by the method according to claim 2, and diagnosing or assisting in diagnosing the subject with rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease if the PAD4 activity is higher than a threshold.

4. The method according to claim 1, 2, or 3, wherein the substrate peptide containing the L-arginine residue has an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].

5. The method according to claim 4, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Met-Asp-Arg-Asp-Gly.

6. The measurement method according to claim 1, wherein PAD is PAD2.

7. The method according to claim 6 for diagnosing Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, or for assisting in the diagnosis of Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, wherein the sample is a biological sample obtained from a subject, the method comprising measuring the PAD2 activity of the biological sample by the method according to claim 6, and diagnosing or assisting in diagnosing that the subject has Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease if the PAD2 activity is higher than a threshold.

8. The substrate peptide containing the L-arginine residue has an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; and the third amino acid residue is L-arginine (Arg). The method according to claim 1, 6, or 7, wherein the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, and the fifth amino acid residue X5 is glycine.

9. The method according to claim 8, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Lys-Gln-Arg-Lys-Gly.

10. The method according to any one of claims 1 to 9, wherein the citrulline oxidase is a modified citrulline oxidase, the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, in the modified citrulline oxidase, one or more amino acids are substituted at positions corresponding to positions 291, 302, 554, or 221, or 502, 479, 305, 310, or 113 of SEQ ID NO: 1, and the modified citrulline oxidase has reduced reactivity to L-arginine and increased activity to L-citrulline compared to the original polypeptide.

11. In the modified citrulline oxidase, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is substituted with glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase; the amino acid at the position corresponding to position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid in the modified citrulline oxidase; the amino acid at the position corresponding to position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at the position corresponding to position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. The method according to claim 10, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine, and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

12. A modified citrulline oxidase, wherein the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, and in the modified citrulline oxidase, one or more amino acids are substituted at positions corresponding to positions 291, 302, 554, or 221, or 502, 479, 305, 310, or 113 of SEQ ID NO: 1, and compared to the original polypeptide, the modified citrulline oxidase has decreased reactivity to L-arginine and increased activity to L-citrulline.

13. In the modified citrulline oxidase, the amino acid at position 291 of SEQ ID NO: 1 is substituted with glutamine; the amino acid at position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine; the amino acid at position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine; the amino acid at position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid; the amino acid at position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. The modified citrulline oxidase according to claim 12, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan, the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine, and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

14. A modified citrulline oxidase according to any one of claims 12 to 13, wherein the polypeptide before modification is a polypeptide derived from the genus Streptomyces.

15. A modified citrulline oxidase according to any one of claims 12 to 14, comprising FAD as a coenzyme.

16. A composition for measuring PAD activity, comprising the modified citrulline oxidase described in any one of claims 12 to 15.

17. The composition according to claim 16, comprising a substrate peptide containing an L-arginine residue having PAD4, and further comprising an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].

18. The composition according to claim 17, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Met-Asp-Arg-Asp-Gly.

19. PAD is PAD2, and furthermore, the amino acid sequence consists of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; and the third amino acid residue is L-arginine (Arg). The composition according to claim 16, comprising a substrate peptide containing an L-arginine residue, wherein the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine, and the fifth amino acid residue X5 is glycine.

20. The composition according to claim 19, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Lys-Gln-Arg-Lys-Gly.

21. The composition according to any one of claims 16 to 20, further comprising peptidase.

22. The composition according to claim 21, comprising multiple types of peptidases.

23. The composition according to claim 22, wherein the plurality of peptidases are selected from the group consisting of (i) a first endopeptidase and a second endopeptidase, (ii) an endopeptidase and an exopeptidase, and (iii) a first exopeptidase and a second exopeptidase.

24. A polynucleotide encoding a modified citrulline oxidase according to any one of claims 12 to 15.

25. A vector comprising the polynucleotide described in claim 24.

26. A host cell comprising the vector according to claim 25.

27. A method for producing modified citrulline oxidase, comprising: (i) culturing host cells according to claim 25 to produce modified citrulline oxidase according to any one of claims 12 to 15; and (ii) obtaining the produced modified citrulline oxidase.

28. A composition for measuring PAD4 activity by enzymatic method, comprising a substrate peptide containing an L-arginine residue having an amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, glycine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid and glycine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, and L-valine; and the fifth amino acid residue X5 is glycine].

29. The composition according to claim 28, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Met-Asp-Arg-Asp-Gly.

30. A composition comprising the composition according to claim 28 or 29, a peptidase, and citrulline oxidase, for the diagnosis of rheumatoid arthritis, fibrosis, cancer, multiple sclerosis, ulcerative colitis, or Alzheimer's disease.

31. Amino acid sequence consisting of X1-X2-Arg-X4-X5 [wherein the formula, the first amino acid residue X1 is selected from the group consisting of L-methionine or L-serine, L-aspartic acid or L-glutamic acid, L-histidine, L-leucine, L-isoleucine, L-valine, or L-lysine; the second amino acid residue X2 is selected from the group consisting of L-aspartic acid, glycine, L-serine, L-tryptophan, L-tyrosine, L-isoleucine, L-valine, L-glutamine, and L-phenylalanine; the third amino acid residue is L-arginine (Arg); the fourth amino acid residue X4 is selected from the group consisting of L-aspartic acid or glycine, L-histidine, L-asparagine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-threonine, and L-valine] A composition for measuring PAD2 activity by enzymatic method, comprising a substrate peptide containing an L-arginine residue, having the fifth amino acid residue X5 being glycine.

32. The composition according to claim 31, wherein the substrate peptide containing the L-arginine residue has the amino acid sequence Lys-Gln-Arg-Lys-Gly.

33. A composition for diagnosing Alzheimer's disease, cancer, multiple sclerosis, or inflammatory bowel disease, comprising the composition according to claim 31 or 32, a peptidase, and citrulline oxidase.

34. The composition according to any one of claims 28 to 33, wherein the citrulline oxidase is a modified citrulline oxidase, the modified citrulline oxidase is based on the original polypeptide, the original polypeptide has 70% or more, 80% or more, or 90% or more amino acid sequence identity with SEQ ID NO: 1, in the modified citrulline oxidase, the amino acid at the position corresponding to position 291 of SEQ ID NO: 1 is substituted with glutamine, and compared to the original polypeptide, the modified citrulline oxidase has decreased reactivity to L-arginine and increased activity to L-citrulline.

35. In the modified citrulline oxidase, further, the amino acid at the position corresponding to position 302 of SEQ ID NO: 1 is substituted with methionine, isoleucine, or leucine in the modified citrulline oxidase; the amino acid at the position corresponding to position 554 of SEQ ID NO: 1 is substituted with methionine, leucine, asparagine, isoleucine, or glutamine in the modified citrulline oxidase; the amino acid at the position corresponding to position 221 of SEQ ID NO: 1 is substituted with aspartic acid or glutamic acid in the modified citrulline oxidase; the amino acid at the position corresponding to position 502 of SEQ ID NO: 1 is substituted with glycine or alanine; the amino acid at the position corresponding to position 479 of SEQ ID NO: 1 is substituted with leucine, alanine, serine, tryptophan, methionine, tyrosine, glutamine, valine, or threonine. The composition according to claim 34, wherein the amino acid at position 305 of SEQ ID NO: 1 is substituted with valine, asparagine, isoleucine, leucine, glutamine, methionine, histidine, threonine, phenylalanine, or tryptophan; the amino acid at position 310 of SEQ ID NO: 1 is substituted with valine, leucine, or methionine; and / or the amino acid at position 113 of SEQ ID NO: 1 is substituted with arginine.

36. A method for quantifying L-citrulline, comprising the step of contacting a sample that may contain L-citrulline with a modified citrulline oxidase according to any one of claims 12 to 15.

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