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Defective mucin-type glycosylation on ?-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases.

ABSTRACT: Deficiency in subunits of the conserved oligomeric Golgi (COG) complex results in pleiotropic defects in glycosylation and causes congenital disorders in humans. Insight regarding the functional consequences of this defective glycosylation and the identity of specific glycoproteins affected is lacking. A chemical glycobiology strategy was adopted to identify the surface glycoproteins most sensitive to altered glycosylation in COG-deficient Chinese hamster ovary (CHO) cells. Following metabolic labeling, an unexpected increase in GalNAz incorporation into several glycoproteins, including ?-dystroglycan (?-DG), was noted in cog1-deficient ldlB cells. Western blotting analysis showed a significantly lower molecular weight for ?-DG in ldlB cells compared with WT CHO cells. The underglycosylated ?-DG molecules on ldlB cells are highly vulnerable to bacterial proteases that co-purify with V. cholerae neuraminidase, leading to rapid removal of the protein from the cell surface. The purified bacterial mucinase StcE can cleave both WT and ldlB ?-DG but did not cause rapid degradation of the fragments, implicating other V. cholerae proteases in the final proteolysis of the fragments. Extending terminal glycosylation on the existing mucin-type glycans of ldlB ?-DG stabilized the resulting fragments, indicating that fragment stability, but not the initial fragmentation of the protein, is influenced by the glycosylation status of the cell. This discovery highlights a functional importance for mucin-type O-glycans found on ?-DG and reinforces a growing role for these glycans as regulators of extracellular proteolysis and protein stability.

SUBMITTER: Yu SH

PROVIDER: S-EPMC6139567 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Defective mucin-type glycosylation on α-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases.

Yu Seok-Ho SH Zhao Peng P Prabhakar Pradeep K PK Sun Tiantian T Beedle Aaron A Boons Geert-Jan GJ Moremen Kelley W KW Wells Lance L Steet Richard R

The Journal of biological chemistry 20180726 37

Deficiency in subunits of the conserved oligomeric Golgi (COG) complex results in pleiotropic defects in glycosylation and causes congenital disorders in humans. Insight regarding the functional consequences of this defective glycosylation and the identity of specific glycoproteins affected is lacking. A chemical glycobiology strategy was adopted to identify the surface glycoproteins most sensitive to altered glycosylation in COG-deficient Chinese hamster ovary (CHO) cells. Following metabolic l ...[more]

PMID: 30049793

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Project description:BACKGROUND & AIMS:Core 1- and core 3-derived mucin-type O-linked oligosaccharides (O-glycans) are major components of the colonic mucus layer. Defective forms of colonic O-glycans, such as the Thomsen-nouveau (Tn) antigen, frequently are observed in patients with ulcerative colitis and colorectal cancer, but it is not clear if they contribute to their pathogenesis. We investigated whether and how impaired O-glycosylation contributes to the development of colitis-associated colorectal cancer using mice lacking intestinal core 1- and core 3-derived O-glycans. METHODS:We generated mice that lack core 1- and core 3-derived intestinal O-glycans (DKO mice) and analyzed them, along with mice that singly lack intestinal epithelial core 1 O-glycans (IEC C1galt1(-/-) mice) or core 3 O-glycans (C3Gnt(-/-) mice). Intestinal tissues were collected at different time points and analyzed for levels of mucin and Tn antigen, development of colitis, and tumor formation using imaging, quantitative polymerase chain reaction, immunoblot, and enzyme-linked immunosorbent assay techniques. We also used cellular and genetic approaches, as well as intestinal microbiota depletion, to identify inflammatory mediators and pathways that contribute to disease in DKO and wild-type littermates (controls). RESULTS:Intestinal tissues from DKO mice contained higher levels of Tn antigen and had more severe spontaneous chronic colitis than tissues from IEC C1galt1(-/-) mice, whereas spontaneous colitis was absent in C3GnT(-/-) and control mice. IEC C1galt1(-/-) mice and DKO mice developed spontaneous colorectal tumors, although the onset of tumors in the DKO mice occurred earlier (age, 8-9 months) than that in IEC C1galt1(-/-) mice (15 months old). Antibiotic depletion of the microbiota did not cause loss of Tn antigen but did reduce the development of colitis and cancer formation in DKO mice. Colon tissues from DKO mice, but not control mice, contained active forms of caspase 1 and increased caspase 11, which were reduced after antibiotic administration. Supernatants from colon tissues of DKO mice contained increased levels of interleukin-1? and interleukin-18, compared with those from control mice. Disruption of the caspase 1 and caspase 11 genes in DKO mice (DKO/Casp1/11(-/-) mice) decreased the development of colitis and cancer, characterized by reduced colonic thickening, hyperplasia, inflammatory infiltrate, and tumors compared with DKO mice. CONCLUSIONS:Impaired expression of O-glycans causes colonic mucus barrier breach and subsequent microbiota-mediated activation of caspase 1-dependent inflammasomes in colonic epithelial cells of mice. These processes could contribute to colitis-associated colon cancer in humans.

Dataset Information

Defective mucin-type glycosylation on ?-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases.

Publications

Defective mucin-type glycosylation on α-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases.

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