Aberrations in O-mannosyl glycan modifications on the protein alpha- dystroglyan, have been associated with loss of function and forms of muscular dystrophy those also manifest significant neurological consequences. This was the first protein on which O-mannosylation and associated glycans were directly identified in the animal kingdom, and has led to the emerging understanding of several new glycosylation biosynthetic pathways. Until now, investigations have relied heavily on the application of genetic analysis and manipulation of animal model systems. The strategy has identified a number of players, but it has limitations in fully elaborating the process at a molecular level, also complicated by the intrinsic microheterogeneity of protein glycosylation in vivo. A chemical biology approach is needed to fully elucidate these glycan structures, the regulation of their synthesis, and the molecular basis of disease. This will entail the use of chemical synthesis to access defined glycopeptides as intermediates in quantifying enzymatic processing, thereby allowing detailed characterization of enzyme activities and decoding of their substrate requirements, coupled with mass spectrometry to analyze glycan structures. This information is critical in fully revealing the structure/function relationships of these glycans, in disease diagnosis, and in rational design of therapeutic strategies. The broader relevance of O- mannosylation and its biosynthetic pathway to other proteins are becoming more apparent, with several recent reports describing additional targets, including its prevalence on cadherins. In this proposal we will focus on the complex O-mannosyl glycan structure critical to the interactions with the extracellular matrix.
The first aim will determine the factors that regulate he action of the recently identified enzyme POMGNT2. This functions at a key step, selecting O-Man residues for commitment to elaborate the core glycan to which the laminin binding structure, that mediates interaction with the extracellular matrix, is attached.
The second aim wil identify the as yet unknown structure that links this core trisaccharide glycan with the distal laminin binding oligosaccharide, and thus gain insight into the activities that assemble it.
The third aim will leverage our glycopeptide synthetic approach to make antibodies to specific structures, providing reagents that offer promise in diagnosis and which will facilitate studies of the broader range of glycoproteins on which this form of glycosylation is found.
A set of congenital muscular dystrophies, termed dystroglycanopathies, has been related to errors in assembly of the carbohydrate modifications on the glycoprotein alpha-dystroglycan, a cell surface protein that plays an important role in organizing tissues through linking cells to the extracellular matrix. The biological processes by which these carbohydrate structures are elaborated and mediate interactions with the extracellular matrix are still being elucidated. Our goal is to use chemical and biochemical tools to fully understand this and provide a rational basis for the design of strategies that will remediate consequences of glycosylation defects, and disease.
