Endoglycosidases that cleave
View Patent ↗Methods and compositions have been described that relate to a newly identified polypeptide family wherein each member has O-glycosidase activity and specified sequence characteristics. This family of enzymes can be used for example for cleaving O-linked glycans and for synthesis of neoglycopeptides or neoglycoproteins.
1. A preparation in a reaction vessel, comprising a recombinant polypeptide and glycerol, the recombinant polypeptide comprising amino acids 1-1155 of SEQ ID NO: 4, wherein the recombinant polypeptide consists of less than 1400 amino acids, and lacks a carbohydrate-binding domain at the C-terminal end.
2. A synthetic vector comprising a nucleic acid that encodes a polypeptide according to claim 1 .
3. A method, comprising:
a) combining in a reaction vessel a eukaryotic glycoprotein or glycopeptide with a purified prokaryotic glycosidase having an amino acid sequence comprising:
i) an amino acid sequence at least 35.2% identical to SEQ ID NO: 2;
ii) SEQ ID NO: 1; and
iii) an FDY amino acid sequence in a central conserved domain wherein the number of amino acids between the FDY and a C-terminal end of the polypeptide is no more than 200 amino acids,
wherein the glycosidase polypeptide consists of less than 1400 amino acids and lacks a carbohydrate-binding domain at the C-terminal end;
b) cleaving a Core 3 (GIcNAcβ1,3GaINAc) O-linked glycan in the glycoprotein or glycopeptide; and
c) separating the glycan products cleaved from the glycoprotein or glycopeptide.
4. The method according to claim 3 , further comprising treating the glycoprotein or glycopeptide with a neuraminidase.
5. The method according to claim 4 , further comprising sequentially or simultaneously treating the glycoprotein or glycopeptide with the neuraminidase and the glycosidase.
6. The method according to claim 3 , wherein the glycosidase is active at 25° C.
7. The method according to claim 3 , wherein the glycosidase is capable of cleaving 100% of the Core 3 O-linked glycans as determined by thin layer chromatography (TLC).
8. The method according to claim 3 , further comprising cleaving the Core 3 O-linked glycans at pH 2.0 to 9.0.
9. The preparation according to claim 1 further comprising neuraminidase.