Glycan structures synthesized by specific glycosyltransferases play functional roles in development, cancer progression, metastasis, immunity and signal transduction in vertebrates. In order to understand new and conserved functions of sugars in biology and cancer, we have developed a panel of well-characterized CHO glycosylation mutants and used them to identify roles for defined glycans in signal transduction by Notch receptors, for investigating the properties of the LARGE glycosyltransferase that is required for functional glycosylation of a-dystroglycan, and in determining the recognition specificity of galectins-1, -3 and -8 for cell surface glycans. These experiments have opened up new avenues of research in functional glycomics. In the next five years we will use the CHO glycosylation mutants to explore key questions in glycobiology and we will expand the panel of CHO glycosylation mutants available for functional glycomics.
In Specific Aim 1 we will use CHO cells and CHO glycosylation mutants to determine how mouse Large, Large2, fukutin and fukutin-related protein synthesize functional glycans on a-dystroglycan and how Large rescues dystroglycanopathies.
In Specific Aim 2 we will use CHO cells and CHO mutants to determine the activity and functions of unannotated """"""""orphan"""""""" glycosyltransferases identified in genome searches. The focus will be on three genes that encode potential GlcNAc-transferases predicted to modify complex N- glycans. The corresponding glycosyltransferase genes will be functionally localized in developmental and signaling pathways by characterizing existing mutants or by antisense techniques in zebrafish.
In Specific Aim 3 CHO cells and a subset of CHO mutants will be transformed by Polyoma middle T antigen and used to determine roles for cell surface Gal in different structural contexts in forming functional lattices that modulate growth factor and cytokine signaling with galectins-1, -3, -8 and -9. A similar cohort of CHO mutants will be used to investigate the novel mechanism of galectin secretion.
Specific Aim 4 is to expand the panel of CHO glycosylation mutants available for experiments in functional glycomics. Selection strategies that preclude the isolation of known mutants have been designed: Mutants will be characterized by lectin resistance and rapid glycomic profiling by MALDI-TOF mass spectrometry. The biochemical and genetic basis of new phenotypes will then be determined.
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