Abstract
α-Linked GalNAc (α-GalNAc) is most notably found at the nonreducing terminus of the blood type-determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans. However, despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few α-N-acetylgalactosaminidases are known. Here, to discover and characterize novel microbial enzymes that hydrolyze α-GalNAc, we screened small-insert libraries containing metagenomic DNA from the human gut microbiome. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that specifically cleaves the initial α-GalNAc from mucin-type O-glycans. This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces.
Highlights
␣-Linked GalNAc (␣-GalNAc) is most notably found at the nonreducing terminus of the blood type– determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans
Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that cleaves the initial ␣-GalNAc from mucin-type O-glycans
In the first screening round, an activated aryl monosaccharide glycoside substrate that releases the highly fluorescent 4-methylumbelliferone (MU) reporter upon cleavage was used to screen for ␣-GalNAcase activity (Fig. 2)
Summary
␣-Linked GalNAc (␣-GalNAc) is most notably found at the nonreducing terminus of the blood type– determining A-antigen and as the initial point of attachment to the peptide backbone in mucin-type O-glycans Despite their ubiquity in saccharolytic microbe-rich environments such as the human gut, relatively few ␣-N-acetylgalactosaminidases are known. Using a simple fluorogenic glycoside substrate, we identified and characterized a glycoside hydrolase 109 (GH109) that is active on blood type A-antigen, along with a new subfamily of glycoside hydrolase 31 (GH31) that cleaves the initial ␣-GalNAc from mucin-type O-glycans This represents a new activity in this GH family and a potentially useful new enzyme class for analysis or modification of O-glycans on protein or cell surfaces. GalNAc is found relatively sparingly in nature but plays two important roles One of these is as the first sugar attached to proteins within mucin-type O-glycans, linked to a serine or threonine residue. This would provide an alternative to the use of polypeptide N-acetylgalactosaminyltransferase in the generation of O-linked glycoproteins
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