Journal
GLYCOBIOLOGY
Volume 22, Issue 2, Pages 235-247Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwr131
Keywords
congenital muscular dystrophy; dystroglycan; dystroglycanopathy; like-glycosyltransferase; neural stem cells
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Funding
- National Institutes of Health [HD060458, NS066582]
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Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of alpha-dystroglycan (alpha-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of alpha-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and alpha-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than alpha-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of alpha-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of alpha-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on alpha-DG but also N-glycans on proteins other than alpha-DG.
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