Hydrolysis of mannose-1-phospho-6-mannose linkage to phospho-6-mannose
First Claim
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1. A method for uncapping a mannose-6-phosphate residue on an oligosaccharide, said method comprisinga) providing said oligosaccharide having a mannose-1-phospho-6-mannose linkage;
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(ii) a VRXE motif, where X is any amino acid other than Pro;
(iii) an X1YQGX2 motif, where X1 is Leu, Ile, Val, Ala, Phe, Tyr or Met, and X2 is Thr, Ser, or Asn; and
(iv) GDXGN, where X can be any amino acid other than Pro, andwherein said mannosidase comprises an amino acid sequence having at least 90% identity to residues 1 to 774 of SEQ ID NO;
50 or an amino acid sequence having at least 95% identity to SEQ ID NO;
50.
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Abstract
Described herein are methods and genetically engineered cells useful for uncapping a mannose-6-phosphate residue on an oligosaccharide.
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Citations
39 Claims
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1. A method for uncapping a mannose-6-phosphate residue on an oligosaccharide, said method comprising
a) providing said oligosaccharide having a mannose-1-phospho-6-mannose linkage; - and
b) contacting said oligosaccharide with a mannosidase capable of hydrolyzing said mannose-1-phospho-6-mannose residue to phospho-6-mannose, wherein said mannosidase is a member of glycosyl hydrolase family 92 and wherein said mannosidase does not contain a catalytic acid residue capable of protonating the anomeric oxygen, and wherein said mannosidase comprises an amino acid sequence having (i) a GVGXXGXGG motif where X is Gly, Ala, Ser, Thr, or Cys;
(ii) a VRXE motif, where X is any amino acid other than Pro;
(iii) an X1YQGX2 motif, where X1 is Leu, Ile, Val, Ala, Phe, Tyr or Met, and X2 is Thr, Ser, or Asn; and
(iv) GDXGN, where X can be any amino acid other than Pro, andwherein said mannosidase comprises an amino acid sequence having at least 90% identity to residues 1 to 774 of SEQ ID NO;
50 or an amino acid sequence having at least 95% identity to SEQ ID NO;
50. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method for uncapping a mannose-6-phosphate residue on an oligosaccharide, said method comprising
a) providing said oligosaccharide having a mannose-1-phospho-6-mannose linkage; - and
b) contacting said oligosaccharide with a mannosidase capable of hydrolyzing said mannose-1-phospho-6-mannose residue to phospho-6-mannose, wherein said mannosidase is a member of glycosyl hydrolase family 92 and wherein said mannosidase does not contain a catalytic acid residue capable of protonating the anomeric oxygen, and wherein for said mannosidase, the three dimensional protein coordinates of the atoms in the amino acid side chains located in the minimal catalytic center fall within 1.5 Å
deviation of the coordinates of the equivalent atoms inFIG. 33 , andwherein said mannosidase comprises an amino acid sequence having at least 90% identity to residues 1 to 774 of SEQ ID NO;
50 or an amino acid sequence having at least 95% identity to SEQ ID NO;
50. - View Dependent Claims (27, 28, 29, 30, 31, 32)
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33. A method for uncapping a mannose-6-phosphate residue on an oligosaccharide, said method comprising
a) providing said oligosaccharide having a mannose-1-phospho-6-mannose linkage; - and
b) contacting said oligosaccharide with a mannosidase capable of hydrolyzing said mannose-1-phospho-6-mannose residue to phospho-6-mannose, wherein said mannosidase is a member of glycosyl hydrolase family 92 and wherein said mannosidase does not contain a catalytic acid residue capable of protonating the anomeric oxygen, and wherein said mannosidase comprises an amino acid sequence having at least the following residues forming the catalytic center;
G71, G72, D355, R405, Q536, N588, Q589, T626, D660 and D662, andwherein said mannosidase comprises an amino acid sequence having at least 90% identity to residues 1 to 774 of SEQ ID NO;
50 or an amino acid sequence having at least 95% identity to SEQ ID NO;
50. - View Dependent Claims (34, 35, 36, 37, 38, 39)
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Specification