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Coat of Many Colors - Specificity of GalNAc Transferase Family of Isoenzymes

Paul Daniel, Earnest James
http://orcid.org/0000-0003-1706-0395
http://rave.ohiolink.edu/etdc/view?acc_num=case1586253427484438

Abstract Details

2020, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
Mucin type O-glycosylation is one of the most common and diverse protein modifications in humans. The process is initiated by a large family of N-acetylgalatosamine transferases (GalNAc-T1 to 20), that transfer the GalNAc sugar onto specific serine (Ser) and threonine (Thr) acceptor residues on target proteins. The underlying molecular mechanisms that govern acceptor Thr/Ser site selection by the GalNAc-T are not well understood. Mutations and abnormal expression of individual GalNAc-T isoenzymes lead to alterations in site-specific O-glycosylation, which have been associated with many cancers and metabolic syndromes. Previous substrate specificity studies have shown that the peptide sequence and the prior O-GalNAc glycosylation on a substrate dictate the acceptor site selection by GalNAc-Ts. Structurally, nearly all GalNAc-Ts contain a catalytic domain and a lectin domain connected by a flexible linker. The enzyme activity is enhanced by the lectin domain recognizing remote prior glycosylation (>5 residues away from the acceptor) in an N-or C-terminal direction, and a small number of transferases show rate enhancement by the catalytic domain recognizing neighboring prior glycosylation (<5 residues away). In this thesis we have begun to show how the acceptor site preferences of GalNAc-T isoenzymes are fine-tuned by a combination of multiple enzyme-substrate interactions, guided by enzyme domain specificities that ultimately results in a defined pattern of O-glycosylation. In chapter 1, we show that GalNAc-Ts have intrinsic preference for acceptor Thr residues over Ser residues independent of the flanking peptide sequences. Interestingly the Thr/Ser rate ratio varies between GalNAc-T subfamilies thus adding to the increasing dimension of GalNAc-T substrate specificity. In chapter 2, we show that GalNAc-T2’s preference for both N- and C-terminal remote prior glycosylation is primarily based on its short flexible linker that provides rotational and translational capacity to the lectin domain. This work exemplifies how a structural feature, very distant from the catalytic domain active site, is capable of tuning the activity and specificity of a GalNAc-T. In chapter 3 and 4, kinetic studies reveal that GalNAc-T4 and -T12 isoenzymes, which modify densely glycosylated substrates, are more active against di-glycopeptide substrates with two prior glycosylated O-GalNAc sites, compared to mono-glycopeptides. Specifically, GalNAc-T4’s highest preference is for the lectin domain recognizing remote glycosylation, whereas GalANc-T12’s preference is for the catalytic domain recognizing neighboring glycosylation. Further, kinetic studies performed on O-GalNAc binding site mutants of GalNAc-T4 and -T12, helped to identify the catalytic domain residues in these isoenzymes that recognize the neighboring glycosylation modulating catalytic efficiency. In chapter 5, we show that FGF23, a phosphate regulating hormone, is specifically glycosylated by GalNAc-T3 at Thr178 and this specificity arises through its preferences for remote prior glycosylation of Thr171. We also show that the clustered charged residues flanking the acceptor Thr178, drastically slows the turnover rate, thereby, modulating the amount of intact FGF23 protected from proteolysis. In chapter 6, we continue these studies to begin to survey other GalNAc-T preferences for densely charged substrates. Finally, in chapter 7, we show that individual GalNAc-T’s preference for Thr vs. Ser prior glycosylated residues (Thr* vs. Ser*) varies by its relative position in N- or C-terminal direction. For example, GalNAc-T2 showed equal activity for Thr* and Ser* at the C-terminal but showed almost no activity for Ser* compared to Thr* at the N-terminal of the substrate. In this we have identified an additional unique feature governing GalNAc-T substrate specificity. Taken together, our studies show that distinct combinations of preferences impart unique specificity to each GalNAc-T isoenzyme, including different preferences for Thr or Ser acceptor residues, surface charge density, multiple prior glycosylation, and whether Thr or Ser residues are prior glycosylated.
Thomas Gerken (Advisor)
Martin Snider (Committee Chair)
William Merrick (Committee Co-Chair)
Vincent Monnier (Committee Member)
Biochemistry
ppGalNAc-Ts; GalNAc-Ts; GalNAc-T; GalNAc transferase; GALNT; Glycosyltransferase; mucin type O-glycosylation; O-glycosylation; GalNAc

Recommended Citations

Citations

  • Paul Daniel, E. J. (2020). Coat of Many Colors - Specificity of GalNAc Transferase Family of Isoenzymes [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1586253427484438

    APA Style (7th edition)

  • Paul Daniel, Earnest James. Coat of Many Colors - Specificity of GalNAc Transferase Family of Isoenzymes . 2020. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1586253427484438.

    MLA Style (8th edition)

  • Paul Daniel, Earnest James. "Coat of Many Colors - Specificity of GalNAc Transferase Family of Isoenzymes ." Doctoral dissertation, Case Western Reserve University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1586253427484438

    Chicago Manual of Style (17th edition)

case1586253427484438
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.