Glycoside Hydrolase Family 89 Alpha-N-Acetylglucosaminidase From Clostridium Perfringens Specifically Acts On Glcnac Alpha 1,4gal Beta 1r At The Non-Reducing Terminus Of O-Glycans In Gastric Mucin

In mammals, alpha-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. alpha-N-Acetylglucosaminidases (alpha GNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal alpha GNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of alpha GNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human alpha GNase, by chemically synthesizing a series of disaccharide substrates containing alpha-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAc alpha 1,4Gal beta 1pMP and GlcNAc alpha 1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAc alpha 1,2Gal beta 1pMP, GlcNAc alpha 1,3Gal beta 1pMP, GlcNAc alpha 1,6Gal beta 1pMP, and GlcNAc alpha 1,4GlcA beta 1pMP substrates, this enzyme may represent a specific glycosidase required for degrading alpha-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAc alpha 1,4Gal beta 1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered alpha-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.