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Abstract
The enzyme POMGNT1 catalyzes the transfer of N-acetylglucosamine in a 2-linkage to O-linked mannose on polypeptides in the cis-Golgi apparatus to generate the core M1 glycan. Recently several proteins have been shown to contain O-Mannose glycans that exist as the simple monosaccharide, core M0, without extension. Given the established promiscuous nature of POMGNT1, we sought to explain this surprising finding of M0 structures on the cadherin family. Our work demonstrates that cadherins that carry non-elaborated M0 structures are resistant to POMGNT1 extension at the protein level, unlike alpha-dystroglycan expressed in a mutant background so as to contain M0 glycans. However, upon reduction, alkylation, and tryptic digestion, the M0-containing glycopeptides from both cadherin family proteins as well as alpha- dystroglycan can be modified by POMGNT1. Given that protein folding occurs in the endoplasmic reticulum of the secretory pathway and POMGNT1 is localized to the cis-Golgi, our experiments suggest that the M0 structures on folded cadherins, unlike alpha-dystroglycan, are not accessible for elaboration by POMGNT1 due to structural constraints.
Loss of POMGNT1, responsible for the generation of the core M1 glycan, is seen in Muscle-Eye- Brain (MEB) Disease, one of the secondary dystroglycanopathies resulting from loss of functional glycosylation of alpha-dystroglycan. However, our laboratory and others have determined the structure of the fully elaborated functional M3 glycan on alpha-dystroglycan and there is no apparent POMGNT1-dependent linkage required. Thus, we wanted to investigate the role of POMGNT1 in the generation of the functional M3 glycan that is defective in multiple forms of congenital muscular dystrophy. Several hypotheses were tested with regard to the role of POMGNT1 in functionally glycosylating alpha-dystroglycan. We found that only full-length, catalytically active POMGNT1 could rescue an MEB cell line, demonstrating that enzyme activity is required. Furthermore, while fully-elaborated M3 glycans are not observed on alpha- dystroglycan expressed in MEB cells, we were able to determine the presence of phosphoribitol- extended phosphotrisaccharide M3 glycans on alpha-dystroglycan. Thus, loss of active, full-length POMGNT1 inhibits late steps in the synthesis of the functional M3 glycan. Therefore, our research has shed light on how M0 structures escape POMGNT1 and how the functional M3 structures require POMGNT1 activity.
Loss of POMGNT1, responsible for the generation of the core M1 glycan, is seen in Muscle-Eye- Brain (MEB) Disease, one of the secondary dystroglycanopathies resulting from loss of functional glycosylation of alpha-dystroglycan. However, our laboratory and others have determined the structure of the fully elaborated functional M3 glycan on alpha-dystroglycan and there is no apparent POMGNT1-dependent linkage required. Thus, we wanted to investigate the role of POMGNT1 in the generation of the functional M3 glycan that is defective in multiple forms of congenital muscular dystrophy. Several hypotheses were tested with regard to the role of POMGNT1 in functionally glycosylating alpha-dystroglycan. We found that only full-length, catalytically active POMGNT1 could rescue an MEB cell line, demonstrating that enzyme activity is required. Furthermore, while fully-elaborated M3 glycans are not observed on alpha- dystroglycan expressed in MEB cells, we were able to determine the presence of phosphoribitol- extended phosphotrisaccharide M3 glycans on alpha-dystroglycan. Thus, loss of active, full-length POMGNT1 inhibits late steps in the synthesis of the functional M3 glycan. Therefore, our research has shed light on how M0 structures escape POMGNT1 and how the functional M3 structures require POMGNT1 activity.