JP2020022440A5 - - Google Patents
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- JP2020022440A5 JP2020022440A5 JP2019135853A JP2019135853A JP2020022440A5 JP 2020022440 A5 JP2020022440 A5 JP 2020022440A5 JP 2019135853 A JP2019135853 A JP 2019135853A JP 2019135853 A JP2019135853 A JP 2019135853A JP 2020022440 A5 JP2020022440 A5 JP 2020022440A5
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- JP
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- Prior art keywords
- endo
- sugar chain
- complex
- type sugar
- amino acid
- Prior art date
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- 235000000346 sugar Nutrition 0.000 claims description 54
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 108090000288 Glycoproteins Proteins 0.000 claims description 8
- 102000003886 Glycoproteins Human genes 0.000 claims description 8
- 102000002068 Glycopeptides Human genes 0.000 claims description 7
- 108010015899 Glycopeptides Proteins 0.000 claims description 7
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 12
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims 8
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims 8
- 102100035149 Cytosolic endo-beta-N-acetylglucosaminidase Human genes 0.000 claims 7
- 101710144190 Endo-beta-N-acetylglucosaminidase Proteins 0.000 claims 7
- 230000002255 enzymatic effect Effects 0.000 claims 6
- 241000894006 Bacteria Species 0.000 claims 5
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 claims 3
- 241001470488 Tannerella Species 0.000 claims 3
- 241000606125 Bacteroides Species 0.000 claims 2
- 241001015906 Muribaculum Species 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 244000005700 microbiome Species 0.000 claims 2
- 150000008163 sugars Chemical class 0.000 claims 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims 1
- 125000003047 N-acetyl group Chemical group 0.000 claims 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims 1
- 235000021310 complex sugar Nutrition 0.000 claims 1
- 229930182830 galactose Natural products 0.000 claims 1
- 229960002442 glucosamine Drugs 0.000 claims 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 6
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 6
- 108010064983 Ovomucin Proteins 0.000 description 6
- 239000007795 chemical reaction product Substances 0.000 description 6
- 108010066476 ribonuclease B Proteins 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000006206 glycosylation reaction Methods 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 229950006780 n-acetylglucosamine Drugs 0.000 description 3
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102000007066 Prostate-Specific Antigen Human genes 0.000 description 2
- 101100315399 Caenorhabditis elegans tsp-14 gene Proteins 0.000 description 1
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 102000002493 N-Acetylglucosaminyltransferases Human genes 0.000 description 1
- 108010093077 N-Acetylglucosaminyltransferases Proteins 0.000 description 1
- 108010061952 Orosomucoid Proteins 0.000 description 1
- 102000012404 Orosomucoid Human genes 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Description
高マンノース型糖鎖が結合しているＲＮａｓｅ Ｂ、おもに２～４本鎖複合型糖鎖が結合しているα１－ＡＧＰ、および糖鎖の非還元末端にＮ－アセチルグルコサミンが露出しているｂｉｓｅｃｔｉｎｇ ＧｌｃＮＡｃ含有複合型糖鎖がおもに結合しているｏｖｏｍｕｃｏｉｄ（各１μｇ）の混合溶液（５０ｍＭクエン酸緩衝液、ｐＨ５．０）に対し、Ｅｎｄｏ－Ｔｓｐ１４５７、Ｅｎｄｏ－Ｔｓｐ１２６３、Ｅｎｄｏ－Ｔｓｐ１００６およびＥｎｄｏ－Ｂａｃ１００８の各酵素（各０．６μｇ）を単独あるいは組み合わせて用い、糖鎖切断反応を実施した（４５℃、２０時間）。反応産物をＳＤＳ－ＰＡＧＥに供した結果を図９に示す。 RNase B bound with high-mannose sugar chains, α1-AGP bound mainly with 2- to 4-chain complex-type sugar chains, and bisecting with N-acetylglucosamine exposed at the non-reducing end of sugar chains. Endo-Tsp14 5 7, Endo-Tsp1263, Endo-Tsp1006 and Endo- Each enzyme of Bac1008 (0.6 μg each) was used singly or in combination to carry out sugar chain cleavage reaction (45° C., 20 hours). FIG. 9 shows the results of subjecting the reaction products to SDS-PAGE.
図９の結果より、例えばこれらの糖タンパク質から糖鎖の非還元末端にＮ－アセチルグルコサミンが露出しているｂｉｓｅｃｔｉｎｇ ＧｌｃＮＡｃ含有複合型糖鎖を含むすべての糖鎖を遊離させるためには、Ｅｎｄｏ－Ｔｓｐ１４５７、Ｅｎｄｏ－Ｔｓｐ１２６３、Ｅｎｄｏ－Ｔｓｐ１００６の組合せ、あるいはＥｎｄｏ－Ｔｓｐ１４５７、Ｅｎｄｏ－Ｔｓｐ１２６３、Ｅｎｄｏ－Ｂａｃ１００８の組合せ、もしくはＥｎｄｏ－Ｔｓｐ１４５７、Ｅｎｄｏ－Ｔｓｐ１２６３、Ｅｎｄｏ－Ｔｓｐ１００６、Ｅｎｄｏ－Ｂａｃ１００８のすべての酵素を用いて糖鎖切断反応を実施すればよい。また、これらの糖タンパク質から糖鎖の非還元末端にＮ－アセチルグルコサミンが露出しているｂｉｓｅｃｔｉｎｇ ＧｌｃＮＡｃ含有複合型糖鎖以外の糖鎖を遊離させるためには、Ｅｎｄｏ－Ｔｓｐ１４５７とＥｎｄｏ－Ｔｓｐ１００６の組合せ、あるいはＥｎｄｏ－Ｔｓｐ１４５７とＥｎｄｏ－Ｂａｃ１００８の組合せの酵素を用いて糖鎖切断反応を実施すればよい。 From the results of FIG. 9, for example, in order to release all sugar chains including bisecting GlcNAc-containing complex-type sugar chains in which N-acetylglucosamine is exposed at the non-reducing end of the sugar chain from these glycoproteins, Endo- The combination of Tsp14 5 7, Endo-Tsp1263, Endo-Tsp1006, or the combination of Endo-Tsp14 5 7, Endo-Tsp1263, Endo-Bac1008, or the combination of Endo-Tsp14 5 7, Endo-Tsp1263, Endo-Tsp100-B, En All enzymes may be used to carry out the sugar chain cleavage reaction. In order to release sugar chains other than bisecting GlcNAc-containing complex-type sugar chains in which N-acetylglucosamine is exposed at the non-reducing end of the sugar chain from these glycoproteins, Endo-Tsp14 5 7 and Endo-Tsp1006 or a combination of Endo-Tsp14 5 7 and Endo-Bac1008 may be used to carry out the glycosylation reaction.
Claims (13)
（ａ）配列表配列番号３のアミノ酸配列からなるタンパク質
（ｂ）配列表配列番号３のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつＥｎｄｏ－Ｔｓｐ１００６酵素活性を有するタンパク質
（ｃ）配列表配列番号３のアミノ酸配列と相同性が９０％以上のアミノ酸配列からなり、かつＥｎｄｏ－Ｔｓｐ１００６酵素活性を有するタンパク質 The claim, wherein the endoglycosidase is Endo-Tsp1006, which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Tannerella, and is a protein of the following (a), (b) or (c): 4. The method of claim 2 or claim 3.
(a) a protein consisting of the amino acid sequence of SEQ ID NO: 3 of the sequence listing; Protein having enzymatic activity (c) A protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing and having Endo-Tsp1006 enzymatic activity
（ａ）配列表配列番号７のアミノ酸配列からなるタンパク質
（ｂ）配列表配列番号７のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつＥｎｄｏ－Ｂａｃ１００８酵素活性を有するタンパク質
（ｃ）配列表配列番号７のアミノ酸配列と相同性が９０％以上のアミノ酸配列からなり、かつＥｎｄｏ－Ｂａｃ１００８酵素活性を有するタンパク質 The claim, wherein the endoglycosidase is Endo-Bac1008, which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Muribaculum, and is a protein of the following (a), (b) or (c): 4. The method of claim 2 or claim 3.
(a) a protein consisting of the amino acid sequence of SEQ ID NO:7 in the sequence listing ; Protein having Endo-Bac 1008 enzyme activity (c) A protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 7 in the sequence listing and having Endo- Bac 1008 enzyme activity
（ａ）配列表配列番号１のアミノ酸配列からなるタンパク質
（ｂ）配列表配列番号１のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつＥｎｄｏ－Ｔｓｐ１２６３酵素活性を有するタンパク質
（ｃ）配列表配列番号１のアミノ酸配列と相同性が９０％以上のアミノ酸配列からなり、かつＥｎｄｏ－Ｔｓｐ１２６３酵素活性を有するタンパク質 The claim, wherein the endoglycosidase is Endo-Tsp1263, which is an endo-β-N-acetylglucosaminidase derived from a bacterium belonging to the genus Tannerella, and is a protein of the following (a), (b) or (c): 4. The method according to 4.
(a) a protein consisting of the amino acid sequence of SEQ ID NO: 1 of the sequence listing; Protein having enzymatic activity (c) A protein consisting of an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO: 1 in the sequence listing and having Endo-Tsp1263 enzymatic activity
（ａ）配列表配列番号９のアミノ酸配列からなるタンパク質
（ｂ）配列表配列番号９のアミノ酸配列において１若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつＥｎｄｏ－Ｂｎｏ１２６３酵素活性を有するタンパク質
（ｃ）配列表配列番号９のアミノ酸配列と相同性が９０％以上のアミノ酸配列からなり、かつＥｎｄｏ－Ｂｎｏ１２６３酵素活性を有するタンパク質 The claim, wherein the endoglycosidase is Endo-Bno1263, which is an endo-β-N-acetylglucosaminidase derived from bacteria belonging to the genus Bacteroides, and is a protein of the following (a), (b) or (c): 4. The method of claim 2 or claim 3.
(a) a protein consisting of the amino acid sequence of SEQ ID NO: 9 of the sequence listing; Protein having enzymatic activity (c) A protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 9 in the Sequence Listing and having Endo- Bno 1263 enzymatic activity
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018141928 | 2018-07-28 | ||
| JP2018141928 | 2018-07-28 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020022440A JP2020022440A (en) | 2020-02-13 |
| JP2020022440A5 true JP2020022440A5 (en) | 2022-08-01 |
Family
ID=69617868
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2019135853A Pending JP2020022440A (en) | 2018-07-28 | 2019-07-24 | Method for separating composite type sugar chain |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2020022440A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW202227479A (en) | 2020-09-02 | 2022-07-16 | 日商第一三共股份有限公司 | NOVEL ENDO-[beta]-N-ACETYLGLUCOSAMINIDASE |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013051608A1 (en) * | 2011-10-03 | 2013-04-11 | 独立行政法人産業技術総合研究所 | Composite sugar chain hydrolase |
| JP6252894B2 (en) * | 2013-10-23 | 2017-12-27 | 国立大学法人九州大学 | Endoglycosidase derived from Coprinuscinereus |
-
2019
- 2019-07-24 JP JP2019135853A patent/JP2020022440A/en active Pending
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