ß1,6GlcNAc-branched N-glycans regulate inflammation and cancer

grantor: University of TorontoGlycosylation of cell-surface and secreted glycoproteins is the most complex type of post-translational modification, consuming a large portion of cellular resources, and may have as broad an impact on metazoan biology as phosphorylation. ß1,6GlcNAc-branched asparagine (N)-linked oligosaccharides, the biosynthesis of which requires the activity of ß1,6N-Acetylglucosaniinyltransferase V (GlcNAc-TV), constitute a subset of structurally heterogeneous glycans found on cell surface and secreted glycoproteins. Increased ß1,6GlcNAc-branching of N-glycans has been linked to cancer progression and metastatic potential. In addition, the expression of GlcNAc-TV is induced by activation of Src and Ras. We first examined the expression pattern of 'Mgat5', the gene encoding GlcNAc-TV, during murine development. GlcNAc-TV transcripts were absent in embryonic tissues at E7, but became expressed throughout the embryo at E9.5, and then progressively restricted to regions of the developing central nervous system and to the epithelia of skin, intestine, kidney, endocrine tissues and respiratory tract. A common feature of cells in basal epithelia and in the cortical neural epithelium is the capacity to migrate, a cellular function, which may require GlcNAc-TV dependent glycoconjugates. To examine the function of GlcNAc-TV dependent glycosylation in murine biology, we generated mice deficient in this enzyme by targeted mutation of the 'Mgat5' locus. Homozygous 'Mgat5' -/- mice were viable, fertile. These mice lacked ß1,6GlcNAc-branched N-glycans, indicating that only one gene encodes GlcNAc-TV activity. GlcNAc-TV deficient mice displayed enhanced hypersensitivity to skin irritants. However, the onset of the inflammatory response was slower, likely due to a reduced rate of leukocyte migration 'in vivo', which was associated with increased adhesion to substratum, measured 'in vitro'. GlcNAc-TV deficient T cells, but not B cells, were hypersensitive to mitogenic stimulation. Our results suggest that ß1,6GlcNAc-branched N-glycans on the T cell surface have a direct role as a negative regulator of T cell response to antigen, thus contributing to shutting down immune responses. In order to study the role of GlcNAc-TV in metastatic breast cancer, we crossed GlcNAc-TV deficient mice with mice transgenic for the polyoma middle T antigen (PyMT) under the regulation of a mammary specific promoter. PyMT is a potent oncogene that interacts and activates Shc, Src and PI3 kinase. Signaling through these pathways increases 'Mgat5' expression, making this a relevant model for studying the function of GlcNAc-TV in cancer. GlcNAc-TV deficiency caused a delay in the development of palpable mammary tumors, the area of which grew at a rate 6.7-fold slower than did tumors of wild type littermates, resulting in 4.5-fold reduction in tumor weight. GlcNAc-TV deficiency also resulted in >95% reduction in the incidence of lung metastases. Our results suggest that GlcNAc-TV activity may enhance the formation and turnover of focal adhesions, thereby increasing signaling via the integrin/PI3 kinase/PKB pathway, and influencing cell migration and metastatic potential.Ph.D