Glycoside Hydrolase Family 89 α-N-acetylglucosaminidase from Clostridium perfringens Specifically Acts on GlcNAcα1,4Galβ1R at the Non-reducing Terminus of O-Glycans in Gastric Mucin

Abstract

In mammals, ␣-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. ␣-N-Acetylglucosaminidases (␣GNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal ␣GNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of ␣GNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human ␣GNase, by chemically synthesizing a series of disaccharide substrates containing ␣-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAc␣1,4Gal␤1pMP and GlcNAc␣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␣1,2Gal␤1pMP, GlcNAc␣1,3Gal␤1pMP, GlcNAc␣1,6Gal␤1pMP, and GlcNAc␣1,4GlcA␤1pMP substrates, this enzyme may represent a specific glycosidase required for degrading ␣-GlcNAccapped 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␣1,4Gal␤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 ␣-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.

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

“…Similar truncation studies that focused solely on CBM32s 2 to 6 revealed one or more of these CBMs to be able to bind mucin [11]. Notably, constructs of CpGH89 lacking the three most C-terminal CBMs had reduced activity on mucin suggesting an important role for the CBMs in substrate recognition.…”

Cloning, characterization, and production of three α‐l‐fucosidases from Clostridium perfringens ATCC 13124

“…Therefore, the 1,3‐1,4‐α‐ l ‐fucosidase activity produced would enable C. perfringens ATCC 13124 to remove α1,3/4‐fucosyl residues. In addition to α‐ l ‐fucosidases, C. perfringens is able to produce a series of other exo‐glycosidases, including sialidases , α‐ N ‐acetylglucosaminidase , and β‐ N ‐acetylhexosaminidase , to release the terminal sialic acid, α‐GlcNAc, and α‐GalNAc from mucin O ‐glycans. Endo‐β‐galactosidases of C. perfringens are able to remove special terminal glyco‐epitopes on mucin O ‐glycans, such as Galα1‐3Gal, GlcNAcα1‐4Gal, and A/B blood groups .…”

“…Clostridium perfringens is a common opportunistic pathogen in human intestine, which can cause gas gangrene, necrotic enteritis, food poisoning diarrhea, and non‐foodborne gastrointestinal infections . This pathogen is able to produce various glycosidases (http://www.cazy.org/) for glycan foraging and some of them have been clone and characterized, including endo‐α‐ N ‐acetylgalactosaminidase , sialidases , α‐ N ‐acetylglucosaminidase , and endo‐β‐galactosidases . There are a few reports up to now about its α‐ l ‐fucosidases.…”

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

“…( Figure S6Aii) These proteoglycans are found on mammalian cells and in mucus (Monzon, Casalino-Matsuda and Forteza, 2006). Also, mucins with similar structures to heparan sulfate/heparin ( a-linked GlcNAC or N-Acetyl-D-glucosamine) are present intestinally and found in PGM (Fujita et al, 2011).…”

“…Indeed, the addition of heparanse III, from the soil bacteria Flavobacterium heparinum, which specifically cleaves unmodified (unsulfated) NAc domains (GlcNAc-GlcUA) and NA/NS domains (GlcNS (N-Sulfo-D-Glucosamine) and GlcNAc) of HS, leads to abrogated phage ES17 binding to cells, meaning ES17 was highly specific for this type of glycan (Murphy et al, 2004;Park et al, 2017). Structurally similar mucins to heparan sulfate (Class III type, a-linked GlcNAc) suggest that this protein may also target these intestinal mucins (Ota et al, 1998;Fujita et al, 2011). This interaction is driven by phage ES17's tail fiber protein, which is not related to the hoc proteins or contain an Ig-like fold as predicted from the BAM model.…”