Full Record

New Search | Similar Records

Author
Title Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases
URL
Publication Date
Degree PhD
Discipline/Department Chemistry
Degree Level doctoral
University/Publisher Case Western Reserve University
Abstract Many proteins of eukaryotic cells are known to be O-glycosylated. Glycoproteins with heavily O-glycosylated mucin domains provide important biological functions in a cell: i.e., protection from pathogens, cell-to-cell adhesion and intracellular protein trafficking. Mucin-type O-glycosylation occurs in the Golgi complex and begins with the transfer of GalNAc from UDP-GalNAc onto Ser/Thr residues of polypeptides. This step is catalyzed by a large family (20) called N-a-acetylgalactosaminyl transferases (ppGalNAc-T’s) and forms the GalNAc-a-O-Ser/Thr product. Subsequent elongation is performed by specific glycosyltransferases, producing a variety of glycans. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both subfamilies possess variable activities against glycopeptide substrates. Structurally, 19 isoforms contain a C-terminal catalytic domain linked via a flexible linker to an N-terminal ricin-like lectin domain. The (glyco)peptide substrate specificities of the ppGalNAc-T transferases and the roles of the catalytic and lectin domains in glycopeptide glycosylation still remain largely unknown. Based on the systematic random peptide approach created by the Gerken Lab, I have determined the glycopeptide substrate specificities of several ppGalNAc-T isoforms. A series of (glyco)peptides were created in order to specifically probe the functions of the catalytic and lectin domains in terms of neighboring (1-5 residues) and remote prior glycosylation (6-17 residues) from an acceptor site, respectfully. Using several glycopeptide-preferring isoforms, glycosylation was observed from -4, -3, -1 and +1 relative to a neighboring GalNAc-O-Thr, which I attributed to specific GalNAc-O-Thr binding at the catalytic domain. The other series of glycopeptides contained a GalNAc-O-Thr near the C- or N- terminus of the substrate to address the directionality preferences of the lectin domain. Results with several peptide- and glycopeptide-preferring isoforms revealed preferences that varied among transferase isoform, where some preferred a C-terminally placed GalNAc-O-Thr, or a N-terminally placed GalNAc-O-Thr and others equally preferred the C-/N- terminally placed GalNAc-O-Thr. These directionality preferences are due to the GalNAc-O-Thr interactions at the lectin domain. Results of these studies revealed for the first time the site-specific glycopeptide glycosylation preferences by some ppGalNAc-T’s and has demonstrated that both domains of the ppGalNAc-T’s have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation.
Subjects/Keywords Biochemistry; Chemistry; Mucin-type O-linked glycosylation; ppGalNAc T; lectin domain
Contributors Gerken, Thomas (Advisor); Lee, Irene (Committee Chair)
Language en
Rights unrestricted ; This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
Country of Publication us
Format application/pdf
Record ID oai:etd.ohiolink.edu:case1464000374
Repository ohiolink
Date Retrieved
Date Indexed 2017-02-20
Grantor Case Western Reserve University

Sample Images

.