Method of liberating o-bonded type sugar chain containing peptides and composition for producing o-bonded type sugar chain containing peptides

Abstract

To provide O-bonded type sugar chain-containing peptides that cannot be produced by conventional O-glycoproteases such as IMPa, StcE, and OgpA.SOLUTION: The above problem can be solved by a method for liberating an O-bonded type sugar chain-containing peptides of the present invention, which includes reacting a glycoprotein or glycopeptide with an O-glycoprotease capable of producing a peptide consisting of the amino acid sequence TPPTPSP and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for releasing an O-linked glycan-containing peptide and a composition for producing an O-linked glycan-containing peptide. According to the present invention, it is possible to obtain an O-linked glycan-containing peptide that has not been obtainable before. [Background technology]

[0002] Glycochains that bind to proteins include N-linked glycans that bind to Asn (asparagine) and O-linked glycans that bind to Ser (serine) and Thr (threonine). It is difficult to predict the glycosylation site of O-linked glycans. In addition, enzymes that can excise O-linked glycans with various structures have not been found, and their structural analysis has lagged behind that of N-linked glycans. Conventionally, glycomics analysis of O-linked glycans has mainly been performed using chemical methods, but there were problems such as the destruction of O-linked glycans depending on the reagent used and insufficient excision of glycans. However, a glycoproteomics method has been developed in which glycopeptides of appropriate size containing glycans are prepared and analyzed by mass spectrometry, rather than by excising glycans from glycoproteins. This method can provide information on the structure and binding sites of glycans. However, glycoproteins such as mucins, which are densely bound with O-linked glycans, can be resistant to proteases, making peptide synthesis difficult with existing peptidases / proteases.

[0003] As shown in Figure 1, there are eight typical core structures in O-linked glycans: the core1 structure in which galactose (Gal) is bound by β1-3 to N-acetylgalactosamine (GalNAc) bound to Ser and Thr, the core2 structure in which N-acetylglucosamine (GlcNAc) is bound by β1-6 to the core1 structure, the core3 structure in which GlcNAc is bound by β1-3 to GalNAc bound to Ser and Thr, the core4 structure in which GlcNAc is bound by β1-6 to the core3 structure, the core5 structure in which GalNAc is bound by α1-3 to GalNAc bound to Ser and Thr, the core6 structure in which GlcNAc is bound by β1-6 to GalNAc bound to Ser and Thr, the core7 structure in which GalNAc is bound by α1-6 to GalNAc bound to Ser and Thr, and the core8 structure in which Gal is bound by α1-3 to GalNAc bound to Ser and Thr.

[0004] For example, IMPa derived from Pseudomonas aeruginosa recognizes O-linked glycans including core1 to core4 structures and glycans of a core1 structure to which sialic acid is bound, and cleaves the peptide bond on the N-terminal side of Ser or Thr to which these O-linked glycans are bound (Non-Patent Documents 1 and 2). StcE derived from enterohemorrhagic Escherichia coli recognizes O-linked glycans such as core1 and core2 structures containing sialic acid, and cleaves the peptide bond on the N-terminal side of Ser / Thr when Ser / Thr is present two residues after Ser or Thr to which these O-linked glycans are bound (Non-Patent Documents 1 and 3). Furthermore, OgpA (OpeRATOR) derived from Akkermansia muciniphila recognizes O-linked glycans such as core1, and cleaves the peptide bond on the N-terminal side of Ser or Thr to which these O-linked glycans are bound (Non-Patent Documents 1 and 4). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Biochemical Journal 2021 (UK) vol. 478, pp. 1585-1603 [Non-Patent Document 2] Analytical Chemistry, 2022 (USA), vol. 94, pp. 1060-1069 [Non-Patent Document 3] "Proceedings of the National Academy of Sciences" 2019 (USA) vol.116, pp.7278-7287 [Non-Patent Document 4] Analytical Chemistry, 2018 (USA), vol. 90, p. 8261-8269 [Non-Patent Document 5] "Carbohydrate Research" 2020 (UK) vol.491, p.107981 [Non-Patent Document 6] Pediatric Nephrology 2018 (Germany) vol.33, p.763-777 [Non-Patent Document 7] The Journal of the American Society of Nephrology, 2021 (USA), vol. 32, p. 2455-2465 [Non-Patent Document 8] Proceedings of the National Academy of Sciences, 2017 (USA), vol. 114, E679-E688 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an O-linked glycan-containing peptide that cannot be produced by conventional O-glycoproteases such as IMPa, StcE, and OgpA. [Means for solving the problem]

[0007] The present inventors conducted extensive research into methods for obtaining O-linked glycan-containing peptides that cannot be produced by conventional O-glycoproteases such as IMPa, StcE, and OgpA, and as a result, they surprisingly found that five O-glycoproteases having specific amino acid sequences can release O-linked glycan-containing peptides from glycoproteins that are different from the O-linked glycan-containing peptides obtained by conventional O-glycoproteases. The present invention is based on these findings. Thus, the present invention provides [1] A method for releasing an O-linked glycan-containing peptide, comprising the step of reacting a glycoprotein or glycopeptide with an O-glycoprotease capable of producing a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; [2] The O-glycoprotease is (1) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; (2) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; (3) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 5, or (b) an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 5 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; A method for releasing an O-linked glycan-containing peptide according to [1], which is a method for releasing an O-linked glycan-containing peptide, comprising: (1) a protein having an amino acid sequence represented by SEQ ID NO: 7; (2) a protein having an amino acid sequence which is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 7 and capable of generating a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; or (3) a protein having an amino acid sequence represented by SEQ ID NO: 8; (4) a protein having an amino acid sequence represented by SEQ ID NO: 7; or (b) a protein having an amino acid sequence which is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 7 and capable of generating a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; or (5) a protein having an amino acid sequence represented by SEQ ID NO: 9; or (b) a protein having an amino acid sequence which is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 9 and capable of generating a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; [3] The method for liberating an O-linked glycan-containing peptide according to [1] or [2], wherein in the step of reacting the O-glycoprotease with a glycoprotein or glycopeptide, one or more other O-glycoproteases are reacted. [4] A composition for producing an O-linked glycan-containing peptide, comprising an O-glycoprotease capable of producing a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; [5] The O-glycoprotease is (1) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; (2) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; (3) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 5, or (b) an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 5 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA; A composition for producing an O-linked glycan-containing peptide according to [4], which is a protein capable of generating a peptide consisting of the amino acid sequence of TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP; (4) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7; or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 7 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP; or (5) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9; or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 9 and capable of generating a peptide consisting of the amino acid sequence of TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP; [6] The composition for producing an O-linked glycan-containing peptide according to [4] or [5], further comprising one or more O-glycoproteases different from the O-glycoprotease. Regarding. Effect of the Invention

[0008] According to the method for releasing an O-linked glycan-containing peptide and the composition for producing an O-linked glycan-containing peptide of the present invention, it is possible to obtain an O-linked glycan-containing peptide that could not be obtained by conventional O-glycoproteases. By analyzing the obtained O-linked glycan-containing peptide, the O-linked glycan of the glycoprotein can be analyzed in detail. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the structures of core 1 to core 8 of an O-linked glycan. [Diagram 2] Photographs showing SDS-PAGE electrophoretic patterns of Psd pep, IMPa, EM529, CK pep2, IMPa homolog, SF531, OgpA, StcE, AM0627, AM0908, and AM1514 expressed in E. coli. [Diagram 3] FIG. 1 shows the structures of O-linked glycan-containing glycopeptides and O-linked glycans derived from the IgA hinge region. The IgA hinge region has six O-linked glycan addition sites, underlined 1 to 6. In the present specification, 11 peptide sequences, [a] to [k], were generated by fragmentation treatment with trypsin and O-glycoprotease, and different glycans were bound to each peptide. In the peptide sequences, the amino acid residues in parentheses are the adjacent amino acid residues before fragmentation treatment. The number following the number indicating each glycopeptide indicates the molecular weight of the glycopeptide. [Figure 4]FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained by treating serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, with Psd pep. [Diagram 5] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained by treating serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which were denatured by reductive alkylation and fragmented with trypsin, with CK pep2. [Figure 6] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when an IMPa homolog was reacted with serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, as substrates. [Figure 7] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when EM529 was applied to serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, as substrates. [Figure 8] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when SF531 was applied to serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, as substrates. [Figure 9] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained by treating serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, with IMPa. [Figure 10]FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when OgpA was allowed to act on colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, as a substrate. [Figure 11] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when StcE was allowed to act on colostrum-derived IgA (cIgA) that had been denatured by reductive alkylation and fragmented with trypsin as a substrate. [Figure 12] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained by treating serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which were denatured by reductive alkylation and fragmented with trypsin, with AM0627. [Figure 13] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained by treating serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which were denatured by reductive alkylation and fragmented with trypsin, with AM0908. [Figure 14] FIG. 1 is a graph showing the results of LC ESI-MS analysis of O-linked glycan-containing peptides obtained when AM1514 was applied to serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA), which had been denatured by reductive alkylation and fragmented with trypsin, as substrates. [Figure 15] FIG. 1 shows the results of HPLC analysis of the reaction products obtained by reacting a labeled glycopeptide containing an O-linked glycan (peptide sequence #VESTVAT, an O-linked glycan is attached to the central T, #, fluorescent group) with Psd pep as a substrate. [Figure 16] FIG. 1 shows the results of HPLC analysis of the reaction products obtained by reacting a labeled glycopeptide containing an O-linked glycan (peptide sequence #VESTVAT, an O-linked glycan is attached to the central T, #, fluorescent group) with CK pep2 as a substrate. [Figure 17]FIG. 1 shows the results of HPLC analysis of the reaction products obtained by reacting an IMPa homolog with a labeled glycopeptide containing an O-linked glycan (peptide sequence #VESTVAT, an O-linked glycan is attached to the central T, #, fluorescent group) as a substrate. [Figure 18] FIG. 1 shows the results of HPLC analysis of the reaction products obtained by reacting EM529 with a labeled glycopeptide containing an O-linked glycan (peptide sequence #VESTVAT; an O-linked glycan is attached to the central T; #: fluorescent group) as a substrate. [Figure 19] FIG. 1 shows the results of HPLC analysis of the reaction products obtained by reacting SF531 with a labeled glycopeptide containing an O-linked glycan (peptide sequence #VESTVAT, an O-linked glycan is attached to the central T, #, fluorescent group) as a substrate. [Figure 20] This is a photograph of SDS-PAGE electrophoresis of the reaction products of the enzyme reactions using glycoproteins (fetuin, asialofetuin, or bovine submaxillary mucin (BSM)) of Psd pep, CK pep2, IMPa homolog, EM529, and SF531 as substrates. Arrows are attached to the main cleavage products, and asterisks indicate the enzyme bands. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [1] Method for isolating O-linked glycan-containing peptides The method for releasing an O-linked glycan-containing peptide of the present invention comprises the step of reacting a glycoprotein or glycopeptide with an O-glycoprotease capable of producing a peptide having the amino acid sequence of TPPTPSP (sequence number 11) and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP (sequence number 12) from trypsin-digested human colostrum-derived IgA.

[0011] O-Glycoprotease The O-glycoprotease used in the present invention recognizes the structure of an O-linked glycan and cleaves peptide bonds near Ser and Thr to which the O-linked glycan is bound. The O-glycoprotease acts on glycoproteins such as mucin to which O-linked glycans are densely bound, and can release glycopeptides containing O-linked glycans. The O-glycoprotease used in the present invention has glycan recognition ability and peptide cleavage activity.

[0012] The O-glycoprotease used in the present invention can generate a peptide consisting of the amino acid sequence of TPPTPSP (SEQ ID NO: 11) and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP (SEQ ID NO: 12) from trypsin-digested human colostrum-derived IgA. The TPPTPSP peptide and the HYTNPSQDVTVPCPVPSTPPTPSP peptide are peptides cleaved from an amino acid sequence (KHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL: SEQ ID NO: 13) containing the hinge region of IgA. When generating a TPPTPSP peptide, the O-glycoprotease used in the present invention recognizes an O-glycan bound to the 19th T in the amino acid sequence containing the hinge region of IgA, cleaves the N-terminal side of the T, and recognizes an O-glycan bound to the 26th S, and cleaves the N-terminal side of the S. In addition, when generating the peptides TPPTPSP and HYTNPSQDVTVPCPVPSTPPTPSP, it recognizes the O-glycan bound to the 26th S in the amino acid sequence containing the hinge region of IgA and cleaves the N-terminal side of that S (note that the space between the first K and the second H has been cleaved by trypsin).

[0013] As shown in FIG. 3, peptides [a] to [k] are generated from the hinge region of IgA by the O-glycoprotease used in the present invention and conventional O-glycoproteases. Among the glycopeptides [a] to [k], the glycopeptides ([a] to [i]) having Ser / Thr on the N-terminus have an O-linked glycan bound to this Ser / Thr, and it is considered that the O-glycoprotease, recognizing this, cleaves the peptide bond on the N-terminus of the glycan-bound Ser / Thr. In addition, the C-terminus of the glycopeptides with peptide sequences [a] to [e] was generated by trypsin digestion, and the C-terminus of the glycopeptides with peptide sequences [f] to [i] was generated by O-glycoprotease digestion (an O-linked glycan bound to the adjacent Ser / Thr was cleaved by the O-glycoprotease, recognizing this). In addition, the glycopeptides with peptide sequences [j] and [k] were generated by trypsin digestion on the N-terminus and by O-glycoprotease digestion on the C-terminus.

[0014] Among these glycopeptides, those with multiple glycosylation sites and two glycans are shown, in the case of glycopeptides with peptide sequences [b], [d], [e], [f], [g], and [h], it is considered that a glycan that is recognized by the O-glycoprotease that produced the glycopeptide is bound to the N-terminal Ser / Thr, and that a glycan that is not recognized by the O-glycoprotease is bound to another glycosylation site, or that a glycan is bound to a site where the peptide cleavage reaction by the O-glycoprotease does not occur even if it is recognized. In addition, in the case of glycopeptide with peptide sequence [k], it is considered that a glycan that is not recognized by the O-glycoprotease that produced the glycopeptide is bound to one of the glycosylation sites, or that a glycan is bound to a site where the peptide cleavage reaction by the O-glycoprotease does not occur even if it is recognized. The O-glycoprotease used in the present invention is particularly capable of producing peptides having diverse sugar chain structures such as [b], [f], or [k]. The peptide of TPPTPSP (SEQ ID NO: 11) is peptide [f], and the peptide of HYTNPSQDVTVPCPVPSTPPTPSP (SEQ ID NO: 12) is peptide [k].

[0015] (Psd pep) One embodiment of the O-glycoprotease includes (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1 and capable of producing a peptide consisting of the amino acid sequence of TPPTPSP (SEQ ID NO: 11) and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA (hereinafter, (a) and (b) may be collectively referred to as Psd pep). Psd pep may be derived from, but is not limited to, a bacterium of the genus Pseudomonas. As shown in Figure 4, Psd pep produces glycopeptides with peptide sequences [a], [b], [c], [h], [j] and [k] from human serum-derived IgA (sIgA) and produces glycopeptides with peptide sequences [a], [b] and [k] from human colostrum-derived IgA (cIgA). In particular, it is able to produce peptides [b] and [k] remarkably.

[0016] (CK pep2) One embodiment of the O-glycoprotease includes (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3 and capable of producing a peptide consisting of the amino acid sequence TPPTPSP and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA (hereinafter, (a) and (b) may be collectively referred to as CK pep2). CK pep2 may be derived from, but is not limited to, bacteria of the genus Chitinimonas. As shown in Figure 5, CK pep2 produces glycopeptides with peptide sequences [a], [b], [d], [e], [h], [j] and [k] from human serum-derived IgA (sIgA) and produces glycopeptides with peptide sequences [a], [b], [h], [j] and [k] from human colostrum-derived IgA (cIgA). In particular, it is able to produce peptides [b], [j] and [k] remarkably.

[0017] (IMPa homologue) One embodiment of the O-glycoprotease includes (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 5, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 5 and capable of producing a peptide consisting of the amino acid sequence TPPTPSP and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA (hereinafter, (a) and (b) may be collectively referred to as IMPa homolog). The IMPa homolog may be, but is not limited to, one derived from a bacterium of the genus Duganella. As shown in Figure 6, the IMPa homolog produces glycopeptides with peptide sequences [a], [b], [h], and [k] from human serum-derived IgA (sIgA), and produces glycopeptides with peptide sequences [a], [b], [h], [j], and [k] from human colostrum-derived IgA (cIgA). In particular, it is able to produce peptides [a], [b], and [k] remarkably.

[0018] (EM529) One embodiment of the O-glycoprotease includes (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 7, or (b) a protein consisting of an amino acid sequence that has an identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 7 and that can generate a peptide consisting of the amino acid sequence TPPTPSP and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA (hereinafter, (a) and (b) may be collectively referred to as EM529). EM529 may be derived from, but is not limited to, bacteria of the genus Elizabethkingia. EM529 produces glycopeptides with peptide sequences [a], [b], [c], [d], [f], [g], and [h] from human serum-derived IgA (sIgA), and produces glycopeptides with peptide sequences [a], [b], [d], [f], [g], [h], and [j] from human colostrum-derived IgA (cIgA), as shown in Figure 7. In particular, it is able to produce peptides [b], [f], and [g] remarkably.

[0019] (SF531) One embodiment of the O-glycoprotease includes (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or (b) a protein consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 9 and capable of producing a peptide consisting of the amino acid sequence TPPTPSP and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA (hereinafter, (a) and (b) may be collectively referred to as SF531). SF531 may be derived from, but is not limited to, a bacterium of the genus Sphingobacterium. As shown in Figure 8, SF531 produces glycopeptides with peptide sequences [a], [b], [c], [d], [f] and [k] from human serum-derived IgA (sIgA) and produces glycopeptides with peptide sequences [a], [b], [d], [f], [g] and [k] from human colostrum-derived IgA (cIgA). In particular, it is able to produce peptides [b], [f] and [k] remarkably.

[0020] Psd pep, CK pep2, IMPa homolog, EM529, and SF531 used in the present invention may be proteins consisting of amino acid sequences having 90% or more identity with the respective amino acid sequences, so long as they have functions equivalent to those of the respective O-glycoproteases. The identity of the amino acid sequence is preferably 93% or more, more preferably 95% or more, more preferably 98% or more, and most preferably 99% or more.

[0021] The amino acid sequence having an identity of 90% or more may be, for example, an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added to each amino acid sequence. The O-glycoprotease polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, so long as the identity is 90% or more. In the present specification, "an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added" means that the amino acid sequence has been modified by amino acid substitution or the like. The number of amino acid modifications is not particularly limited as long as the amino acid identity is 90% or more, and is, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, or 1 to 40, preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 2. An example of a modified amino acid sequence of an O-glycoprotease that can be used in the present invention is preferably an amino acid sequence having one or several (preferably 1, 2, 3, or 4) conservative substitutions in its amino acids.

[0022] The O-glycoprotease of the present invention includes the O-glycoprotease of the present invention even if multiple amino acids at the N-terminus and / or multiple amino acids at the C-terminus are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, as long as the effect of the present invention can be obtained. The "amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added" or the "amino acid sequence having an identity of 90% or more" in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9 is substituted in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, or 9, and this substitution in the amino acid sequence is a conservative substitution that maintains the function of the O-glycoprotease of the present invention. In other words, "conservative substitution" means a substitution that does not lose the excellent effect of the O-glycoprotease of the present invention. That is, even when the insertion, substitution, deletion, or addition occurs, it is a substitution that can generate a peptide consisting of the amino acid sequence of TPPTPSP (SEQ ID NO: 11) and / or a peptide consisting of the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP (SEQ ID NO: 12) from trypsin-digested human colostrum-derived IgA. Specifically, it means replacing an amino acid residue with another chemically similar amino acid residue. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known in the art for each amino acid. Examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, lysine, etc., while negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc.

[0023] In the method of releasing an O-linked glycan-containing peptide of the present invention, although not limited thereto, preferably, in the step of reacting an O-glycoprotease with a glycoprotein or glycopeptide, another one or more O-glycoproteases are reacted. The other one or more O-glycoproteases may be one or more O-glycoproteases selected from Psd pep, CK pep2, IMPa homolog, EM529, and SF531 used in the present invention. In addition, they may be O-glycoproteases such as IMPa, StcE, OgpA, AM0627, AM0908, or AM1514 that have been reported in the past (Non-Patent Document 1). Each glycopeptidase recognizes a different glycan structure and cleaves a different amino acid, and by using two or more glycopeptidases in combination, it is possible to obtain a larger number of O-linked glycan-containing peptides from a glycoprotein or glycopeptide. Therefore, by analyzing a large number of obtained O-linked glycan-containing peptides, detailed glycan structure data of the glycoprotein or glycopeptide can be obtained.

[0024] In the method of releasing an O-linked glycan-containing peptide of the present invention, an optimal buffer solution and reaction temperature can be selected depending on each enzyme. For example, buffer solutions include Tris-HCl buffer, phosphate buffer, MOPS buffer, etc. The reaction temperature is preferably 25 to 65°C, more preferably 35 to 55°C. The reaction time is not limited, but is 10 minutes to 48 hours, preferably 30 minutes to 24 hours.

[0025] 《Analysis method》 By analyzing the O-linked glycan-containing peptide obtained by the method of releasing an O-linked glycan-containing peptide of the present invention, the O-linked glycan of a glycoprotein or glycopeptide can be analyzed in detail. The method of analyzing the O-linked glycan-containing peptide is not limited, and examples thereof include mass spectrometry, liquid chromatography, and chemical techniques. By such analyses, the binding site and glycan structure of the O-linked glycan can be clarified. Furthermore, if the released O-linked glycan-containing glycopeptide is collected and fractionated and purified, it can be used as a source of O-linked glycans with various structures and as a source of peptides having specific sequences.

[0026] [2] Composition for producing O-linked glycan-containing peptide The composition for producing an O-linked glycan-containing peptide of the present invention contains an O-glycoprotease capable of producing a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPPTPSP from trypsin-digested human colostrum-derived IgA. The composition for producing an O-linked glycan-containing peptide of the present invention can be used in a method for releasing the O-linked glycan-containing peptide, a method for analyzing the O-linked glycan-containing peptide, etc.

[0027] The composition for producing an O-linked glycan-containing peptide of the present invention can contain, in addition to the O-glycoprotease, a carrier or other components, etc. As other components, for example, water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oil or fatty acid ester, isotonicity agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers can be optionally used.

[0028] The O-glycoprotease can be any O-glycoprotease described in the above "Method for releasing O-linked glycan-containing peptide" without being limited thereto. EXAMPLES

[0029] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0030] <<Production Examples 1 to 5 and Comparative Production Examples 1 to 6>> In this example, Psd pep (Production Example 1), CK pep2 (Production Example 2), IMPa homolog (Production Example 3), EM529 (Production Example 4), and SF531 (Production Example 5) were obtained. The nucleic acid of SEQ ID NO: 2 (gene of Psd pep), the nucleic acid of SEQ ID NO: 4 (gene of CK pep2), the nucleic acid of SEQ ID NO: 6 (gene of IMPa homolog), the nucleic acid of SEQ ID NO: 8 (gene of EM529) and the nucleic acid of SEQ ID NO: 10 (gene of SF531) were artificially synthesized, and each of them was inserted into a pET21a vector (Novagen) to construct an expression vector for expressing each enzyme in E. coli as a fusion protein with a histidine tag (His×6) consisting of 6 histidine residues. As Comparative Production Examples 1 to 6, expression vectors were similarly constructed for IMPa (Comparative Production Example 1), OgpA (Comparative Production Example 2), StcE (Comparative Production Example 3), AM0627 (Comparative Production Example 4), AM0908 (Comparative Production Example 5) and AM1514 (Comparative Production Example 6) based on known sequence information.

[0031] The resulting expression vector was used to transform Escherichia coli BL21 (DE3) strain, and the transformant was cultured in 100 mL of LB medium supplemented with 50 μg / mL ampicillin at 37 ° C for 3 hours. Next, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to 0.1 mM, and the transformant was cultured at 37 ° C for 3.5 hours in the case of AM0908, at 25 ° C for 3.5 hours in the case of OgpA, and at 15 ° C for 21 to 22 hours in the case of other enzymes. Then, the cells were collected by centrifugation, suspended in 5 mL of phosphate-buffered saline (PBS), and disrupted by an ultrasonic disrupter. The surfactant Triton X-100 was added to 1%, and the mixture was shaken at room temperature for 30 minutes, and the supernatant was collected by centrifugation. An appropriate amount of Ni-IMAC Profinity (BIO-RAD) was added to the supernatant, and the mixture was stirred at 4 ° C for 16 hours, and each enzyme was adsorbed to Ni-IMAC Profinity.

[0032] The Ni-IMAC Profinity was collected by centrifugation and then thoroughly washed with washing buffer (20 mM phosphate buffer [pH 7.4], 500 mM NaCl, 0.01% Tween 20), and then 0.3 mL of elution buffer (washing buffer + 300 mM imidazole) was added and shaken at 16°C for 10 minutes to elute the target enzyme adsorbed to Ni-IMAC Profinity. This procedure was repeated again, and the collected eluates were combined and concentrated by ultrafiltration using VIVASPIN500 (molecular weight cutoff 3,000, Sartorius) to obtain a purified enzyme preparation. Figure 2 shows the migration patterns of each purified enzyme by SDS-polyacrylamide gel electrophoresis (PAGE).

[0033] Example 1 In this example, Psd pep obtained in Production Example 1 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved.

[0034] sIgA (Sigma-Aldrich) and cIgA (Sigma-Aldrich) were denatured by cleavage of disulfide bonds and alkylation of cysteine ​​residues (reductive alkylation), and further fragmented by trypsin digestion. 10 μg of these denatured fragmented IgA were added with 1 μg of Psd pep and mixed in 50 mM Tris-HCl buffer (pH 7.5) (reaction volume 10 μL) and reacted at 37°C for 24 hours. The reaction products were analyzed by LC ESI-MS.

[0035] As shown in Figure 4, glycopeptides with peptide sequences [a], [b], [c], [h], [j], and [k] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], and [k] were detected from cIgA. A total of 21 types of glycopeptides were detected in both. Among these, glycopeptides such as peptide No. 8, 12, 47, 51, 53, 54, and 55 were frequently detected from sIgA, and glycopeptides such as peptide No. 5, 8, 11, 12, 13, 50, 51, 52, 53, 54, and 55 were frequently detected from cIgA. On the other hand, glycopeptides with peptide sequences [d], [e], [f], [g], and [i] were not detected at all in either sIgA or cIgA. In other words, glycopeptide species containing core 2-type glycans with peptide sequence [k] (peptide No. 50-55) were frequently detected.

[0036] Example 2 In this example, CK pep2 obtained in Production Example 2 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that CK pep2 was used instead of Psd pep.

[0037] As shown in Figure 5, glycopeptides with peptide sequences [a], [b], [d], [e], [h], [j], and [k] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], [h], [j], and [k] were detected from cIgA. A total of 34 types of glycopeptides were detected in both. Of these, glycopeptides such as peptide No. 8, 12, 46, and 47 were frequently detected from sIgA, and glycopeptides such as peptide No. 5, 8, 11, 12, 13, 45, 46, 47, 50, and 54 were frequently detected from cIgA. On the other hand, glycopeptides with peptide sequences [c], [f], [g], and [i] were not detected at all in either sIgA or cIgA. In other words, glycopeptide species containing core 2-type glycans with peptide sequence [k] (peptide No. 50-55) were frequently detected.

[0038] Example 3 In this example, the IMPa homolog obtained in Production Example 3 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that the IMPa homolog was used instead of Psd pep.

[0039] As shown in Figure 6, glycopeptides with peptide sequences [a], [b], [h], and [k] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], [h], [j], and [k] were detected from cIgA. A total of 20 types of glycopeptides were detected in both. Of these, glycopeptides such as peptide No. 3, 12, 51, 52, 53, 54, and 55 were frequently detected from sIgA, and glycopeptides No. 3, 5, 12, 13, 46, 50, 51, 52, 53, 54, and 55 were frequently detected from cIgA. On the other hand, glycopeptides with peptide sequences [c], [d], [e], [f], [g], and [i] were not detected at all in either sIgA or cIgA. In other words, glycopeptide species (peptides No. 50 to 55) containing core 2-type glycans with peptide sequence [k] were frequently detected.

[0040] Example 4 In this example, EM529 obtained in Production Example 4 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that EM529 was used instead of Psd pep.

[0041] As shown in Figure 7, glycopeptides with peptide sequences [a], [b], [c], [d], [f], [g], and [h] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], [d], [f], [g], [h], and [j] were detected from cIgA. A total of 33 types of glycopeptides were detected in both. Of these, glycopeptides such as peptide No. 8, 12, 38, and 39 were frequently detected in sIgA, and glycopeptides such as peptide No. 5, 8, 12, and 13 were frequently detected in cIgA. On the other hand, glycopeptides with peptide sequences [e], [i], and [k] were not detected at all in either sIgA or cIgA. In other words, glycopeptide species with peptide sequences [f] and [g] (peptide No. 29 to 39) were frequently detected.

[0042] Example 5 In this example, SF531 obtained in Production Example 5 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that SF531 was used instead of Psd pep.

[0043] As shown in Figure 8, glycopeptides with peptide sequences [a], [b], [c], [d], [f], and [k] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], [d], [f], [g], and [k] were detected from cIgA. A total of 28 types of glycopeptides were detected in both. Of these, glycopeptides such as peptide No. 8, 11, 12, and 55 were frequently detected in sIgA, and glycopeptides such as peptide No. 5, 8, 12, 13, 30, 32, 33, and 35 were frequently detected in cIgA. On the other hand, glycopeptides with peptide sequences [e], [h], [i], and [j] were not detected at all in either sIgA or cIgA. In other words, glycopeptide species with peptide sequence [f] (peptide No. 29 to 37) were frequently detected.

[0044] Comparative Example 1 In this comparative example, IMPa obtained in Comparative Production Example 1 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that IMPa was used instead of Psd pep.

[0045] As shown in Figure 9, glycopeptides with peptide sequences [a], [b], [c], [d], [e], [h], [i], and [j] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], [h], [i], and [j] were detected from cIgA. A total of 36 types of glycopeptides were detected in both. Of these, glycopeptides such as peptide No. 3, 8, 45, 46, and 47 were frequently detected in sIgA, and glycopeptide No. 46 was frequently detected in cIgA. On the other hand, glycopeptides with peptide sequences [f], [g], and [k] were not detected at all in either sIgA or cIgA.

[0046] Comparative Example 2 In this comparative example, OgpA obtained in Comparative Production Example 2 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that OgpA and cIgA were used instead of Psd pep.

[0047] As shown in Figure 10, four glycopeptides, peptide Nos. 2, 17, 45, and 47, were detected from cIgA. On the other hand, no glycopeptides having the peptide sequences [b], [e], [f], [g], [h], [i], or [k] were detected from cIgA.

[0048] Comparative Example 3 In this comparative example, an O-linked glycan-containing peptide in the hinge region of immunoglobulin A was obtained using StcE obtained in Comparative Production Example 3. Specifically, an O-linked glycan-containing peptide present in the hinge region of colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that StcE and cIgA were used instead of Psd pep.

[0049] As shown in Figure 11, four glycopeptides, peptide Nos. 8, 12, 17, and 22, were detected from cIgA. On the other hand, no glycopeptides having the peptide sequences [a], [c], [f], [g], [h], [i], [j], or [k] were detected from cIgA.

[0050] Comparative Example 4 In this comparative example, AM0627 obtained in Comparative Production Example 4 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that AM0627 was used instead of Psd pep.

[0051] As shown in Figure 12, glycopeptides with peptide sequences [b], [c], [e], [h], and [j] were detected from sIgA, and glycopeptides with peptide sequences [a], [b], and [j] were detected from cIgA. A total of 20 types of glycopeptides were detected from both. On the other hand, no glycopeptides with peptide sequences [d], [f], [g], [i], or [k] were detected in either sIgA or cIgA.

[0052] Comparative Example 5 In this comparative example, an O-linked glycan-containing peptide in the hinge region of immunoglobulin A was obtained using AM0908 obtained in Comparative Production Example 5. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that AM0908 was used instead of Psd pep.

[0053] As shown in Figure 13, only one type of glycopeptide (α2,3 sialyl core 1 bound at two sites) of peptide No. 9 having the peptide sequence [b] was detected from sIgA, but no glycopeptides were detected from cIgA.

[0054] Comparative Example 6 In this comparative example, AM1514 obtained in Comparative Production Example 6 was used to obtain an O-linked glycan-containing peptide in the hinge region of immunoglobulin A. Specifically, the O-linked glycan-containing peptide present in the hinge region of serum-derived IgA (sIgA) and colostrum-derived IgA (cIgA) was cleaved. The procedure of Example 1 was repeated, except that AM1514 was used instead of Psd pep.

[0055] As shown in Figure 14, two types of glycopeptides were detected from sIgA: a glycopeptide of peptide No. 3 having the peptide sequence [a] (GalNAc bound to the N-terminal Thr) and a glycopeptide of peptide No. 9 having the peptide sequence [b] (α2,3sialylcore1 bound to two sites), but neither glycopeptide was detected from cIgA.

[0056] Example 6 In this example, the cleavage activity of O-linked glycan-containing glycopeptides was examined using Psd pep obtained in Production Example 1. A glycopeptide library was prepared in which different O-linked glycans were bound to peptides with the same amino acid sequence, and the glycan recognition ability and cleavage activity of Psd pep were examined using this as a substrate. A glycopeptide derived from κ-casein glycomacropeptide (peptide sequence VESTVAT, disialylcore1 glycan bound to the central T) was prepared by extracting proteose peptone from commercially available whey protein powder, hydrolyzing it with thermolysin, and purifying it with liquid chromatography (Non-Patent Document 5). A labeled glycopeptide was then prepared by introducing a fluorescent group (#) to the N-terminus of this glycopeptide. This glycopeptide was then treated with sialidase to prepare glycopeptides bound to core1, α2,3sialylcore1, and α2,6sialylcore1 glycans. The glycopeptide with the core1 glycan prepared here was treated with core2 synthase to prepare a glycopeptide bound to a core2 glycan. The cleavage activity of Psd pep was measured using these labeled glycopeptides as substrates. 1 μg of the enzyme and 0.1 mM labeled glycopeptide were mixed in 50 mM Tris-HCl buffer (pH 7.5) (reaction volume 10 μL), reacted at 37°C for 24 hours, and the reaction products were analyzed by HPLC.

[0057] As shown in Figure 15, in all glycopeptides bearing O-linked glycans, core1, α2,3sialylcore1, α2,6sialylcore1, core2, and disialylcore1, cleavage occurred at the N-terminus of the glycan-bound T, and the cleavage product #VES was detected.

[0058] Example 7 In this example, the cleavage activity against O-linked glycan-containing glycopeptides was examined using CK pep2 obtained in Production Example 2. The procedure of Example 6 was repeated, except that CK pep2 was used instead of Psd pep.

[0059] As shown in Figure 16, for glycopeptides with O-linked glycans of any of core1, α2,3sialylcore1, α2,6sialylcore1, core2, and disialylcore1, cleavage occurred at the N-terminus of the glycan-bound T and at the N-terminus of the S next to the glycan-bound T. In the case of core1 and α2,3sialylcore1 glycans, cleavage at the N-terminus of the S next to the glycan-bound T was more likely to occur than cleavage at the N-terminus of the glycan-bound T, and #VE was more frequently detected than #VES. On the other hand, in the case of α2,6sialylcore1, core2, and disialylcore1 glycans, cleavage at the N-terminus of the glycan-bound T was sometimes more likely to occur.

[0060] Example 8 In this example, the cleavage activity against O-linked glycan-containing glycopeptides was examined using the IMPa homolog obtained in Production Example 3. The procedure of Example 6 was repeated, except that the IMPa homolog was used instead of Psd pep.

[0061] As shown in Figure 17, cleavage occurred in glycopeptides with any of the O-linked glycans bound to core1, α2,3sialylcore1, α2,6sialylcore1, core2, and disialylcore1, but the cleavage pattern differed depending on the substrate. In the cases of core1, α2,3sialylcore1, and core2 glycans, cleavage occurred at the N-terminus of the glycan-bound T and the N-terminus of the S next to the glycan-bound T, but the amount of cleavage product was small when the glycan was core2, possibly due to poor glycan recognition ability or cleavage efficiency. On the other hand, in the cases of α2,6sialylcore1 and disialylcore1 glycans, cleavage occurred at the N-terminus of the glycan-bound T.

[0062] Example 9 In this example, the cleavage activity against O-linked glycan-containing glycopeptides was examined using EM529 obtained in Production Example 4. The procedure of Example 6 was repeated, except that EM529 was used instead of Psd pep.

[0063] As shown in Figure 18, when the glycan was core1 or α2,3sialylcore1, cleavage occurred mainly at the N-terminus of the glycan-bound T, but some cleavage also occurred at the N-terminus of the S next to the glycan-bound T. On the other hand, when the glycan was α2,6sialylcore1, core2, or disialylcore1, almost no cleavage occurred.

[0064] Example 10 In this example, the cleavage activity against O-linked glycan-containing glycopeptides was examined using SF531 obtained in Production Example 5. The procedure of Example 6 was repeated, except that SF531 was used instead of Psd pep.

[0065] As shown in Figure 19, when the glycan was core1, cleavage occurred at the N-terminus of the glycan-bound T, but when the glycan was α2,3sialylcore1, cleavage occurred at the N-terminus of the glycan-bound T and at the N-terminus of the S next to the glycan-bound T. On the other hand, when the glycan was α2,6sialylcore1, core2, or disialylcore1, almost no cleavage occurred.

[0066] 《Analysis example》 In this analytical example, fetuin, asialofetuin, and bovine submaxillary mucin (BSM) were cleaved using Psd pep, CK pep2, IMPa homolog, EM529, and SF531. 0.5 to 2 μg of each enzyme was mixed with 4 μg of undenatured fetuin or asialofetuin, or 52 μg of BSM in 50 mM Tris-HCl buffer (pH 7.0 or pH 7.5) (reaction volume 10 μL) and reacted at 37°C for 24 hours, after which the cleavage status of the glycoprotein was analyzed by SDS-PAGE. Both Psd pep and CK pep2 cleaved Fetuin, Asialofetuin, and BSM (Figure 20). In addition, IMPa homologs and EM529 also cleaved Fetuin, Asialofetuin, and BSM (Figure 20). EM529 had low cleavage activity against Fetuin, but showed a cleavage pattern similar to that of CK pep2. SF531 cleaved Asialofetuin and BSM (Figure 20). [Industrial Applicability]

[0067] The method for releasing an O-linked glycan-containing peptide and the composition for producing an O-linked glycan-containing peptide of the present invention can be used in the analysis of O-linked glycans of glycoproteins or glycopeptides.

Claims

1. The method includes reacting a glycoprotein or glycopeptide with an O-glycoprotease capable of producing a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTPTPSP from trypsin-digested human colostrum-derived IgA, A method for releasing O-linked glycan-containing peptides.

2. The O-glycoprotease is (1) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and capable of producing a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (2) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (3) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO:5, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:5, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (4) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7, or (b) a protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 7 and can generate a peptide having the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide having the amino acid sequence HYTNPSQDVTVPCPVPSTPTPSP; or (5) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:9, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; The method for releasing an O-linked glycan-containing peptide according to claim 1,

3. The method for liberating an O-linked glycan-containing peptide according to claim 1 or 2, wherein in the step of reacting the O-glycoprotease with a glycoprotein or glycopeptide, one or more additional O-glycoproteases are reacted.

4. The present invention includes an O-glycoprotease capable of generating a peptide having the amino acid sequence of TPPTPSP and / or a peptide having the amino acid sequence of HYTNPSQDVTVPCPVPSTTPPTPSP from trypsin-digested human colostrum-derived IgA. A composition for producing an O-linked glycan-containing peptide.

5. The O-glycoprotease is (1) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, and capable of producing a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (2) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 3, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (3) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO:5, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:5, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; (4) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 7, or (b) a protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 7 and can generate a peptide having the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide having the amino acid sequence HYTNPSQDVTVPCPVPSTPTPSP; or (5) (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9, or (b) a protein consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:9, and capable of generating a peptide consisting of the amino acid sequence TPPTPSP from trypsin-digested human colostrum-derived IgA and / or a peptide consisting of the amino acid sequence HYTNPSQDVTVPCPVPSTTPPTPSP; The composition for producing an O-linked glycan-containing peptide according to claim 4,

6. The composition for producing an O-linked glycan-containing peptide according to claim 4 or 5, further comprising one or more O-glycoproteases different from the O-glycoprotease.

Images