Abstract 1054 Determining the peptide and glycopeptide substrate specificities of the polypeptide glycosylating transferase GalNAc-T7

Mucin-type protein O-glycosylation, also known as O-glycosylation, is initiated by a large family of UDP-GalNAc:polypeptide α-Nacetylgalactosaminyltransferases (GalNAc-Ts) that transfers α-GalNAc from UDP-GalNAc onto serine and threonine residues of polypeptide acceptors. Many members of the GalNAc-Ts family, including GalNAc-T7, are associated with cancer, including cervical and prostate cancers. Using randomized peptide substrates, the Gerken lab has shown the GalNAc-Ts have both unique and overlapping peptide substrate preferences. In addition, GalNAc-Ts have unique specificities for prior glycosylated serine and threonine residues of glycopeptide substrates, ranging from 1 to 17 residues from the acceptor site. These activities are referred to as short- and long-range prior glycosylation activities. These different glycosylation properties give individual GalNAc-Ts their unique specificities. Despite this, specific protein targets for each of the GalNAc-Ts members, including GalNAc-T7, remain largely unknown. GalNAc-T7 is unique by displaying low glycosylation efficiency against serine or threonine residues on non-glycosylated protein acceptors, while it readily adds GalNAc onto peptides that already have a prior GalNAc residue directly positioned C-terminal of the Threonine or Serine acceptor. In this study, we have obtained the random peptide substrate motifs for GalNAc-T7 for comparison to the previously determined patterns for GalNAc-T10, which belongs to the same subfamily as GalNAc-T7. Studies against Ser-O-GalNAc and Thr-O-GalNAc acceptors have further revealed that Thr-O-GalNAc glycopeptides are more efficiently glycosylated, revealing different kinetic Km and similar Vmax values. Studies are in progress attempting to understand the conformational and binding differences between Ser-O-GalNAc and Thr-O-GalNAc substrates. These studies will help us understand how GalNAc-T7 chooses its protein targets. We acknowledge Dr. Ramon Hurtado-Guerrero at the University of Zaragoza for supplying us with hGalNAc-T7 and the NIH Grant R01GM113534 to TAG.