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Leslie Revoredo Thesis Final.pdf (24.38 MB)

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Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases

Revoredo, Leslie
http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374

Abstract Details

2016, Doctor of Philosophy, Case Western Reserve University, Chemistry.
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.
Thomas Gerken (Advisor)
Irene Lee (Committee Chair)
Michael Zagorski (Committee Member)
Paul Carey (Committee Member)
Michael Harris (Committee Member)
417 p.
Biochemistry; Chemistry
Mucin-type O-linked glycosylation; ppGalNAc T; lectin domain

Recommended Citations

Citations

  • Revoredo, L. (2016). Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374

    APA Style (7th edition)

  • Revoredo, Leslie. Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases. 2016. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374.

    MLA Style (8th edition)

  • Revoredo, Leslie. "Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases." Doctoral dissertation, Case Western Reserve University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374

    Chicago Manual of Style (17th edition)

case1464000374
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© 2016, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.