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 α-dystroglycan (α-DG), was noted in cog1-deficient ldlB cells. Western blotting analysis showed a significantly lower molecular weight for α-DG in ldlB cells compared with WT CHO cells. The underglycosylated α-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 α-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 α-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 α-DG and reinforces a growing role for these glycans as regulators of extracellular proteolysis and protein stability.
Highlights
Deficiency in subunits of the conserved oligomeric Golgi (COG) complex results in pleiotropic defects in glycosylation and causes congenital disorders in humans
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
Altered glycosylation of dystroglycan in COG-deficient cells that co-purify with V. cholerae neuraminidase (Fig. 1)
Summary
Increased labeling of specific glycoproteins with GalNAz in ldlB CHO cells We envisioned that metabolic labeling of cells with Ac4ManNAz or Ac4GalNAz could be an effective way to identify changes in the glycosylation status and abundance of cellsurface glycoproteins in the glycosylation-deficient cells. When labeling was performed using C1GalT1, ST3Gal, UDP-galactose, and CMP-Neu5Ac together, the MW shift of the ␣-DG band increased further (Fig. 7A) This result indicates that ␣-DG in ldlB cells bears Tn antigen (Ser/Thr–O-GalNAc), which cannot accept sialic acid directly by ST3Gal without prior galactosylation. These shifts noted are relatively small compared with the large difference in molecular weight between ldlB and WT derived ␣-DG, which can be interpreted as either the substantial loss of O-glycan occupancy on this glycoprotein or the inefficiency of the enzymatic labeling at the cell surface. B, ldlB cells labeled with C1GalT1 alone or in combination with ST3Gal were treated with or without V. cholerae neuraminidase, followed by SDS-PAGE and Western blotting with the anti-DG (core) antibody. These findings point to a role for the mucin-type glycans in stabilizing the fragments produced by mucinase digestion but not in preventing the initial fragmentation by this enzyme
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