Tmem119 fragment peptide and method for producing the same

Abstract

To identify a peptide that is a small molecular fragment of Tmem119 after its cleavage, and to enable production of the peptide.SOLUTION: A method for producing a Tmem119 fragment peptide includes a step of introducing an expression vector comprising the nucleotide sequence of SEQ ID NO:8 into a host cell capable of gene expression by the expression vector, a step of culturing the host cell under conditions that allow gene expression, and a step of recovering a peptide comprising any one of the amino acid sequences of SEQ ID NOs:2 to 7 from the host cell.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a Tmem119 fragment peptide and a method for producing the same.

Background Art

[0002] Currently, many diseases are known. Among them, cancer, Alzheimer's disease, etc. are known as diseases that significantly reduce the quality of life. Their causes are the accumulation of tumor cells, amyloid plaques, etc. that are unnecessary for the living body. Conventional imaging diagnostic techniques such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI), and Positron Emission Tomography (PET) are effective in detecting aggregates such as tumors and amyloid. However, it is difficult to detect them when the accumulation amount is small. Therefore, even if tumors or aggregates are discovered by the above-mentioned conventional techniques, the disease state may already have progressed and it may be too late.

[0003] So far, it has been known that microglia, known as nerve-immune cells, play a central role in the recognition and phagocytosis of aggregates such as amyloid that are unnecessary for the living body, and are also known to be related to inflammation in the brain. Tmem119 is known as a type I transmembrane protein molecule that is specifically expressed in microglia, and research has been conducted on the possibility of using Tmem119 as a marker for microglia (see, for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] From the above facts, it is conceivable that Tmem119 can be used as a marker for brain inflammation. That is, it is possible to estimate the degree of inflammation in the brain from the expression level of Tmem119. As described above, Tmem119 is a type I transmembrane protein molecule, but so far, nothing is known about the metabolism of Tmem119, and there is no report of detecting molecular fragments of Tmem119. That is, the cleavage site in Tmem119 and the structure of the fragments obtained by cleavage are still unknown. Since the molecules generated by cleavage are considered to be released into the blood and increased with the expression of the full-length Tmem119 molecule when microglia increase and become active, if the metabolism of Tmem119 is clarified, it is considered possible to use the molecular fragments whose release into the blood is assumed by cleavage as markers for brain inflammation. In order to make the molecular fragments available as biomarkers for brain inflammation, it is necessary to identify the amino acid sequences of the molecular fragments. If the molecular fragments can be produced after identifying them, it may be possible to produce antibodies and the like for detecting the molecular fragments.

[0006] The present invention has been made in view of the above problems, and an object thereof is to identify a peptide that is a molecular fragment after cleavage of Tmem119 and to enable the production of the peptide.

Means for Solving the Problems

[0007] In order to achieve the above object, the present inventors have completed the present invention by specifically identifying the cleavage site in the transmembrane region of Tmem119 as a result of intensive research.

[0008] Specifically, the Tmem119 fragment peptide according to the present invention is characterized by containing any one of the amino acid sequences of SEQ ID NOs: 2 to 7.

[0009] The Tmem119 fragment peptide according to the present invention preferably contains a tag molecule.

[0010] In addition, the method for producing the Tmem119 fragment peptide according to the present invention includes a step of introducing an expression vector containing the nucleotide sequence of SEQ ID NO: 8 into a host cell capable of gene expression by the expression vector, a step of culturing the host cell under conditions allowing the gene expression, and a step of recovering the above peptide from the host cell.

[0011] In the method for producing the Tmem119 fragment peptide according to the present invention, it is preferable that the expression vector further contains a nucleotide sequence encoding a tag molecule.

Advantages of the Invention

[0012] According to the Tmem119 fragment peptide and the method for producing the same according to the present invention, it is possible to obtain a small molecular fragment of Tmem119 after cleavage, which can be used as a biomarker for intracerebral inflammation in neurological diseases, and an antibody or the like for detecting the small molecular fragment can be prepared using the same, which can be beneficial for the diagnosis of neurological diseases.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0014] Hereinafter, modes for carrying out the present invention will be described. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its use.

[0015] One embodiment of the present invention is a Tmem119 fragment peptide containing any one of the amino acid sequences of SEQ ID NOs: 2 to 7.

[0016] Tmem119 is known as a type I transmembrane protein that is specifically expressed in microglia. In particular, Tmem119 is a protein with a full length of 283 amino acids, and the 97th to 117th amino acid sequences are known as transmembrane regions (transmembrane domains). Since Tmem119 is specifically expressed in microglia as described above, it has been used as a marker for microglia. In addition, microglia are known to play a central role in the recognition and phagocytosis of aggregates unnecessary for the living body such as amyloid, and are also known to be related to inflammation in the brain. Therefore, it is conceivable that Tmem119, which is a marker for microglia, could be a marker for brain inflammation. The amino acid sequence of Tmem119 is as shown in SEQ ID NO: 1 below. SEQ ID NO: 1: MVSAAAPSLLILLLLLLGSVPATDARSVPLKATFLEDVAGSGEAEGSSASSPSLPPPWTPALSPTSMGPQPITLGGPSPPTNFLDGIVDFFRQYVMLIAVVGSLAFLLMFIVCAAVITRQKQKASAYYPSSFPKKKYVDQSDRAGGPRAFSEVPDRAPDSRPEEALDSSRQLQADILAATQNLKSPTRAALGGGDGARMVEGRGAEEEEKGSQEGDQEVQGHGVPVETPEAQEEPCSGVLEGAVVAGEGQGELEGSLLLAQEAQGPVGPPESPCACSSVHPSV

[0017] As described above, the metabolism of Tmem119 has not been clarified so far, and the cleavage site of Tmem119 and the structure of the fragments obtained by cleavage remain unknown. However, as a result of intensive research, the present inventors have identified the cleavage site of Tmem119 by γ-secretase as described in the Examples below. Specifically, Tmem119 is cleaved at the transmembrane domain, specifically, between leucine 97 and isoleucine 98, between isoleucine 98 and alanine 99, between alanine 99 and valine 100, between valine 100 and valine 101, between serine 103 and leucine 104, or between leucine 107 and leucine 108 in its amino acid sequence. When Tmem119 is cleaved at the above cleavage site, it is considered that the small molecular fragment on the N-terminal side located extracellularly is released extracellularly. In this case, the amino acid at the C-terminal of the small molecular fragment is leucine 97, isoleucine 98, alanine 99, valine 100, serine 103, or leucine 107.

[0018] Since the transmembrane domain of Tmem119 is cleaved by γ-secretase, the portion located intracellularly (intracellular domain: ICD) is not necessary for examining the cleavage site and generating small molecular fragments, so a part of it may be omitted (deleted).

[0019] As shown in FIG. 1, type I transmembrane proteins such as Tmem119 often undergo cleavage of the outer portion (N-terminal side) of the cell membrane by sheddase in addition to cleavage of the transmembrane domain by γ-secretase during the metabolic process. Therefore, Tmem119 may also be cleaved by sheddase, but even in that case, the molecular fragment of Tmem119 (Tmem119 fragment peptide) has the cleavage site by γ-secretase at the C-terminus and has at least about several tens of amino acids. Therefore, the molecular fragment of Tmem119 includes, for example, the sequence from the 85th aspartic acid to the 97th leucine (SEQ ID NO: 2) of the amino acid sequence of Tmem119, up to the 98th isoleucine (SEQ ID NO: 3), up to the 99th alanine (SEQ ID NO: 4), up to the 100th valine (SEQ ID NO: 5), up to the 103rd serine (SEQ ID NO: 6), and up to the 107th leucine (SEQ ID NO: 7) of the amino acid sequence at least at the C-terminus. SEQ ID NO: 2: DGIVDFFRQYVML SEQ ID NO: 3: DGIVDFFRQYVMLI SEQ ID NO: 4: DGIVDFFRQYVMLIA SEQ ID NO: 5: DGIVDFFRQYVMLIAV SEQ ID NO: 6: DGIVDFFRQYVMLIAVVGS SEQ ID NO: 7: DGIVDFFRQYVMLIAVVGSLAFL

[0020] Since the amino acid sequence on the C-terminal side of the Tmem119 fragment peptide generated by cleavage by γ-secretase has been identified, based on this information, a detection molecule such as an antibody that recognizes the C-terminus of the Tmem119 fragment peptide can be manufactured, and by doing so, the fragment peptide can be used as a biomarker for detecting brain inflammation.

[0021] The Tmem119 fragment peptide in this embodiment is not limited in length as long as it contains the above sequence at the C-terminus, but is at most 107 amino acids. Further, the Tmem119 fragment peptide in this embodiment may contain mutations such as substitution, addition, or deletion of at least 10 or less, preferably 5 or less, more preferably 2 or less of the amino acids. Also, a part of the amino acids may be modified. Further, in this embodiment, a tag molecule for detection may be added to a predetermined position such as the N-terminus of the Tmem119 fragment peptide. The tag molecule to be used is not particularly limited as long as it is commonly used in the technical field, and for example, a Flag (registered trademark) tag, His tag, HA tag, Myc tag, etc. can be used.

[0022] Next, another embodiment of the present invention is a method for producing a Tmem119 fragment peptide, comprising the steps of introducing an expression vector containing the nucleotide sequence of SEQ ID NO: 8 into a host cell capable of gene expression by the expression vector, culturing the host cell under conditions capable of gene expression, and recovering the above peptide from the host cell.

[0023] In this embodiment, the nucleotide sequence of SEQ ID NO: 8 is a nucleotide sequence encoding full-length Tmem119. The sequence is as follows. Sequence number 8: atggtttcggcggcagcccccagcctcctcatccttctgttgctgctcctggggtctgtgcctgctaccgacgcccgctctgtgcccctgaaggccacgttcctggaggatgtggcgggtagtggggaggccgagggctcgtcggcctcctccccgagcctcccgccaccctggaccccggccctcagccccacatcgatggggccccagcccataaccctggggggcccatcaccccccaccaacttcctggatgggatagtggacttcttccgccagtacgtgatgctgattgctgtggtgggctccctggcctttctgctgatgttcatcgtctgtgccgcggtcatcacccggcagaagcagaaggcctcggcctattacccatcgtccttccccaagaagaagtacgtggaccagagtgaccgggccgggggcccccgggccttcagtgaggtccccgacagagcccccgacagcaggcccgaggaagccctggattcctcccggcagctccaggccgacatcttggccgccacccagaacctcaagtcccccaccagggctgcactgggcggtggggacggagccaggatggtggagggcaggggcgcagaggaagaggagaagggcagccaggagggggaccaggaagtccagggacatggggtcccagtggagacaccagaggcgcaggaggagccgtgctcaggggtccttgagggggctgtggtggccggtgagggccaaggggagctggaagggtctctcttgttagcccaggaagcccagggaccagtgggtccccccgaaagcccctgtgcttgcagcagtgtccaccccagtgtctaa

[0024] In this embodiment, the expression vector is not particularly limited as long as it can express the nucleotide sequence encoding the full-length Tmem119, and may include control sequences such as a promoter that are suitable for normal expression of the full-length Tmem119 in the host cell into which the vector is introduced. Such control sequences can be appropriately selected by those skilled in the art according to conditions such as the host cell to be used.

[0025] In this embodiment, cells commonly used in the art can be used as the host cell capable of gene expression by the expression vector. For example, immortalized cultured cells such as HEK293 cells can be used, but are not limited thereto.

[0026] In this embodiment, when the host cell of the expression vector is an immortalized cultured cell, transfection techniques commonly used in the art can be used to introduce the expression vector into the host cell. As transfection techniques, for example, chemical means such as the lipofection method and the calcium phosphate coprecipitation method, biological means using adenovirus, etc., and physical means such as the electroporation method can be used, but are not limited thereto. Also, when Escherichia coli is used as the host cell, the expression vector can be introduced using conventional methods such as heat shock using competent cells.

[0027] In this embodiment, the culture of the host cell after gene introduction is carried out under conditions suitable for the expression of the gene introduced into the host cell. Such conditions can be appropriately selected by those skilled in the art. For example, when immortalized cultured cells are used, a medium such as RPMI-1640 medium or Dulbecco's modified Eagle's medium (DMEM) supplemented with serum components such as fetal bovine serum (FBS) as necessary can be used for culturing in an environment of 5% CO2 and 37°C. When Escherichia coli is used, media commonly used in culturing such as SOC medium and LB medium can be used.

[0028] In this embodiment, the method for recovering the Tmem119 peptide from the host cell into which the above expression vector has been introduced can utilize the means for recovering a desired protein from cells commonly used in the art. In particular, since the Tmem119 peptide is present in the cell membrane, the means for recovering proteins from the cell membrane can be utilized. For example, after recovering the host cells into a tube or the like, the cell membrane can be solubilized using a predetermined lysis solution to recover the protein. In addition, it is also possible to produce Tmem119 in an in vitro system using Escherichia coli as shown in the following examples. Here, in order to be able to separate and recover only Tmem119, for example, in the above expression vector, it is preferable to design it to express a protein in which a tag molecule is bound to full-length Tmem119. As the tag molecule, as described above, for example, a Flag (registered trademark) tag, His tag, HA tag, Myc tag, etc. can be used. By expressing Tmem119 to which such a tag molecule is bound in the host cell, only Tmem119 can be separated and recovered using a binding molecule such as an antibody that specifically binds to the tag molecule. At this time, since Tmem119 is cleaved intracellularly, both full-length Tmem119 and the cleaved molecule will be recovered, but since their molecular weights are different, for example, by using SDS-PAGE or the like, they can be easily separated from each other. Also, it can be easily purified by using chromatography or the like. Therefore, it is possible to easily recover only the Tmem119 fragment peptide. It becomes possible to produce the Tmem119 fragment peptide as described above.

Example

[0029] Examples for explaining in detail the Tmem119 fragment peptide and its production method according to the present invention are shown below.

[0030] (Purification of Tmem119 recombinant protein) Competent cells (E. coli BL21 RIL strain: Agilinet, #230245) were prepared and transformed into the competent cells using pET Tmem119, which is a Tmem119 expression vector, by a conventional method. Note that pET Tmem119 was prepared by commissioning GenScript. pET Tmem119 has been codon-optimized and is configured to contain a Flag tag on the N-terminal side and a His tag on the C-terminal side of Tmem119, respectively. The sequence of pET Tmem119 is as shown in SEQ ID NO: 9 below. cttccgccaatatgtgatgctgattgcggtagttggttccctggcttttttgctgatgtttatcgtgtgcgccgcagttattacccgtcaaaagcagaaggcgagcgcatattacccgtctagctttccgaaaaagaaatacgtggaccagtcggatcgtgcgggtggcccacgtgcgttcagcgaggttccggatcgcgctctcgagcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat

[0031] 40 mL of sonication buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA) was added to the obtained transformant, and pipetted to suspend. Then, the suspension was transferred to a tube for sonicator, and sonication was performed using an ultrasonic homogenizer (Branson, Model 250A, 101-063-838) (Duty cycle: 40%, Output Control: 7, Timer: 10 minutes). Then, centrifugation was performed at 18,000 G for 10 minutes at 4°C. 40 mL of sonication buffer was added again to the obtained precipitate, and pipetted to suspend. Sonication was performed on the suspension under the same conditions as above. Then, centrifugation was performed at 18,000 G for 10 minutes at 4°C, and 10 mL of Urea lysis buffer (20 mM Tris-HCl pH 8.5, 6 M Urea, 1% Triton X100, 1% SDS, 1 mM CaCl2, 100 mM NaCl) was added to the precipitate and pipetted. 20 μL of PI mix EDTA (cOmplete) was added to the obtained suspension, and shaken overnight at 4°C.

[0032] The material shaken overnight at 4°C the day before was centrifuged at 18,000 G for 15 minutes at 4°C and transferred to a new 50 mL tube. 30 mL of binding buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl) and 500 μL of Ni-NTA agarose (Quiagen) were added thereto, and the mixture was shaken at room temperature for 30 minutes. The solution after shaking was passed through a column (Bio-Rad, #7311550) for packing. The column was washed successively with the following three washing buffers (1) to (3). (1) 500 μL of 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 1% Triton X-100, (2) 2 mL of 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 0.2% SDS, (3) 500 μL of 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 0.2% SDS, 20 mM imidazole. Next, elution was performed using 500 μL of elution buffer 1 (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 0.2% SDS, 100 mM imidazole) and elution buffer 2 (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 0.2% SDS, 500 mM imidazole).

[0033] Thereafter, after electrophoresis of the obtained eluate, Coomassie staining was performed to confirm the purification degree of the substrate.

[0034] (Recovery of cell membrane of HEK293 cells) To obtain γ-secretase present in the membrane fraction of HEK293 cells, HEK293 cells cultured in a 10 cm dish by a conventional method were prepared and collected in a 50 mL tube. 5 mL of the following hypotonic buffer was added to the 50 mL tube, resuspended, and allowed to stand on ice for 30 minutes. [hypotonic buffer] · 1.5 M sodium citrate salt pH 6.4: 50 μL · 1 M DTT: 50 μL · 0.5 M EDTA: 10 μL · 25× Roche protease inhibitor EDTA+: 200 μL ·H2O: 4690 μL

[0035] Subsequently, the above suspension was placed in liquid nitrogen for 5 minutes to freeze it. Subsequently, it was placed in a water bath for 10 minutes to thaw and then left standing on ice. Then, it was centrifuged at 2500 G for 20 minutes at 4°C, and the supernatant was transferred to a new 15 mL tube, and 450 μL of 65% glycerol (w / w) was added thereto and mixed well. The mixed solution was divided into 6 portions and placed in ultracentrifuge tubes, and ultracentrifuged at 55000 rpm for 1 hour at 4°C. The supernatant was aspirated with an aspirator, and the obtained cell membrane was stored at -80°C.

[0036] (In Vitro γ-Secretase Assay) 50 μL of 1% Chapso Lysis buffer (150 mM citric acid pH 6.4, 1% Chapso, 1×PI Roch EDTA+) was added to the recovered HEK293 cell membrane, and after resuspending well, it was left standing on ice for 30 minutes. Then, it was ultracentrifuged at 55000 rpm for 1 hour at 4°C, and the supernatant containing γ-secretase was collected in a new tube. The required number of PCR tubes were prepared, and 9.6 μL of Pre mix (H2O: 5.85 μL, 1.5 M sodium citrate pH 6.4: 0.75 μL, 25×PI Roche EDTA+: 0.5 μL, the above cell membrane lysate: 2.5 μL) was dispensed into each tube. 0.2 μL of the above purified substrate was added to each tube. 0.2 μL of γ-secretase inhibitor (L-685, 458 ([(2R,4R,5S)-2-benzyl-5-(t-butyloxycarbonylamino)-4-hydroxy-6-phenylhexanoyl]-L-leucyl-L-phenylalanine amide: 4394-v, Peptide Institute INC.)) or DMSO was added to each tube, and then shaken overnight at 37°C to obtain samples.

[0037] (SDS-PAGE and Western Blot) For the samples prepared the day before, 5 μL of 3×Urea sample buffer was added to each sample, and SDS-PAGE was performed using tricine gel. Subsequently, the gel was transferred to a nitrocellulose membrane, boiled in PBS for 5 minutes, and then blocked with 0.2% iblock dissolved in TBS-T for 30 minutes. After shaking overnight at 4°C in a solution containing anti-His antibody and anti-Flag antibody, photographs were taken with AI600 to detect specific bands. The results are shown in Figure 2.

[0038] (Mass spectrometry) Immunoprecipitation was performed by adding anti-His antibody and anti-Flag antibody to the sample and shaking at 4°C for 1 hour. Centrifugation was carried out at 21500×g for 1 minute at 4°C, the supernatant was aspirated, 1 mL of IP-MS buffer (10 mM Tris-HCl, 140 mM NaCl, 5 mM EDTA, 0.1% N-octylglycopyranoside) was added, followed by centrifugation at 21500×g for 1 minute at 4°C, and the supernatant was aspirated. This operation was repeated once more. Then, 1 mL of MilliQ was added, followed by centrifugation at 21500×g for 1 minute at 4°C, the supernatant was aspirated, and this operation was repeated once more. MilliQ was directly taken from a plastic container. Finally, all the moisture was aspirated with a syringe equipped with a needle (NIPRO Flowmax 30G×1 / 2, 01-134). Subsequently, CHCA (α-cyano-4-hydroxycinnamic acid) was added to 500 μL of TWA (50% acetonitrile, 2.5% TFA (trifluoroacetic acid)), and the mixture was shaken at 37°C for 10 minutes. 12 μL of the obtained CHCA / TWA was added to the immunoprecipitated sample, and centrifugation was carried out at 15000 rpm for 1 minute at room temperature. The obtained sample was spotted on a MALDI Target plate (MSP96 Target ground steel BC (Bruker)) at 1 μL per spot, spotted again after drying, and spotted a total of 3 times. After complete drying, peaks were observed in LP (Linear mode) and RP (Reflection mode) by matrix-assisted laser desorption ionization (MALDI) TOF MS. The results are shown in Figure 3.

[0039] (Results of Western blot) As shown in the photograph above Figure 2, under conditions without a γ-secretase inhibitor, a band was detected by an anti-His tag antibody that is thought to be an intracellular domain (ICD) generated by cleavage of Tmem119 around 6 kDa. On the other hand, under conditions containing a γ-secretase inhibitor, that band was not detected. Also, when stained with an anti-Flag tag antibody, under conditions without a γ-secretase inhibitor, bands thought to be β-peptide (Tmem119β) generated by cleavage of Tmem119 were seen around 6 kDa and at a lower position. However, under conditions containing a γ-secretase inhibitor, no bands were seen. In any case, a band of the C-terminal fragment (CTF) of Tmem119 that was not cleaved by γ-secretase was detected around 17 kDa. From the above results, it was suggested that Tmem119 undergoes cleavage by γ-secretase and that there are multiple cleavage sites.

[0040] (Results of mass spectrometry) As shown in the upper graph of Figure 3, as a result of mass spectrometry, six peaks (indicated by ▽1 to 6) were seen in the region of 2700 - 3800 m / z, and these peaks corresponded to the Flag-side fragments of Tmem119 cleaved at the portions indicated by ▽1 to 6 shown below the graph. That is, it was found that the small molecule fragments of Tmem119 were cleaved at the portions where the C-terminus is leucine at position 97, isoleucine at position 98, alanine at position 99, valine at position 100, serine at position 103, and leucine at position 107.

Claims

1. A Tmem119 fragment peptide comprising any of the amino acid sequences of SEQ ID NOs: 2 to 7.

2. The Tmem119 fragment peptide of claim 1 , comprising a tag molecule.

3. Introducing an expression vector containing the nucleotide sequence of SEQ ID NO:8 into a host cell capable of gene expression by the expression vector; culturing said host cells under conditions allowing expression of said gene; A method for producing a Tmem119 fragment peptide, comprising the step of recovering the peptide described in claim 1 or 2 from the host cell.

4. The method for producing a Tmem119 fragment peptide according to claim 3 , wherein the expression vector further comprises a nucleotide sequence encoding a tag molecule.

Images