Glycoproteins containing heavily O-glycosylated mucin domains play important biological roles protecting cell surfaces, modulating cell-cell interactions, targeting of proteins, regulating the inflammatory and immune responses, and in tumorogenisis and metastasis. A number of tumor antigens are mucin-like glycoproteins. O-glycosylated domains play critical roles that depend on their extended structures and ability to be decorated by an array of glycan structures. A family of ppGalNAc transferases adds the first sugar, a- GalNAc, to the peptide core while subsequent transferases elongate the glycan chain by adding sugars to GalNAc. Recent studies suggest that the O-glycan structures can vary in a peptide sequence dependent manner. The mechanisms behind this modulation of O-glycosylation is poorly understood, although both peptide sequence and neighboring group glycosylation effects may be important. It is the aim of this work to characterize in detail the peptide substrate specificities of the family of ppGalNAc transferases and the Core 1, Core 3 and sialyl transferases which add sugars (beta-Gal (1-3), beta-GIcNAc(1-3) and alpha-NeuNAc (2-6) respectively) to the GalNAc.
In Aim 1 the site specific glycosylation kinetics of a series of ppGalNAc transferases against several high molecular weight apo-mucin domains and smaller random peptide substrates will be determined. Experimental apo-mucin glycosylation kinetics will be fit to a kinetic model incorporating neighboring glycosylation and sequence context effects.
In Aim 2 the role of the ricin-like lectin domain found in all ppGalNAc transferases and the effects of small molecule competitors on ppGaINAc transferase site specific glycosylation will be investigated.
In Aim 3 the site specific glycosylation kinetics of the transferases that add to the C3 or C6 positions of the peptide linked GalNAc will be determined and kinetically modeled as above. As a result of this work we will have obtained a sound understanding of the role of peptide sequence and local environment on the modulation of the initial steps of O-glycan biosynthesis. This will lead to a better understanding of the O-glycosylation of biologically important molecules such as CD8, CD45, IgA1, PSGL-1 and the tumor mucin antigens MUC1 and CA125, molecules whose biological properties are known to depend on their O-glycosylation state. Fundamental tools for the rational prediction of site specific O-glycan structures will result from these studies.
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