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Dual functional POGases Encompassing O-glycanase and Adhesin Activities

ABSTRACT: Mucin-type O-glycans on glycoproteins play many pivotal roles in biological processes, making O-glycosylation of biotherapeutics a potential critical quality attribute. Defining the structurefunction relationship of O-glycans is hindered by the lack of enzymatic tools to release all complex O-glycans from glycoproteins. Furthermore, glycans on host cells often serve as ligands for lectins/adhesins on bacteria for bacterium-host interactions, which are essential for the colonization of commensal bacteria and attachment/invasion of pathogens. More emerging adhesins require identification and characterization. To identify endo-α-Nacetylgalactosaminidases (O-glycanases, GH101) with broad substrate specificities, termed Peptide:O-Glycosidase (POGase), we screened putative candidates from 46 different bacteria with fluorogenic α2,3sialylCore 1 substrates, identified five POGase orthologs, and characterized one that releases sialylated complex O-glycans from glycopeptides, glycoproteins and biotherapeutics. Three unique peptide motifs differentiate the POGase existing in phylum Actinomycetota from known O-glycanases. Furthermore, while the POGases contain the GH101 domain, other domains at the C-terminus are required for efficient O-glycanase activity and possess binding activity to major glycans commonly decorating epithelial cells. The dual function POGases, which encompass O-glycanase and adhesin activities, will shed light on O-glycomics, quality assessment of O-glycoprotein drugs, microbiology, and pathogenesis of diseases that may lead to novel therapeutics.

INSTRUMENT(S): ultraflex, Synapt MS

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Bettinger

LAB HEAD: Tongzhong Ju

PROVIDER: PXD051707 | Pride | 2025-02-25

REPOSITORIES: pride

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			Action	DRS
	CD24_23sialylcore1.raw.zip	Raw
	CD24_23sialylcore1_N4.raw.zip	Raw
	CD24_CORE1.zip	Other
	CD24_CORE1_N4.zip	Other
	MUC5Ac_23sialylcore1_N4_dda.raw.zip	Raw

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Project description:Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS 2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

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Dual functional POGases Encompassing O-glycanase and Adhesin Activities

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