Glycoproteins containing heavily O-glycosylated mucin domains play important biological roles including protecting epithelial cell surfaces, modulating cell-cell interactions and regulating the inflammatory and immune responses. They also play important roles in tumorigenesis and metastasis and are common tumor antigens. The O-glycosylated domains in these glycoproteins are required for their proper biological function, principally because of their extended protease resistant structures imparted by the O-glycans. A large family (>20 members) of ppGalNAc transferases initiates the first step of O-glycosylation by adding 1- GalNAc to Ser or Thr residues of the peptide core. Subsequent transferases elongate the glycan chain by sequentially adding sugars to peptide linked GalNAc. Recently mutations in a number of ppGalNAc Ts have been associated with patients with colon cancer. Since the colonic mucin (MUC2) knock-out mouse readily develops colon cancer, we propose that the mutated h-ppGalNAc Ts fail to properly glycosylate h-MUC2, resulting in a colonic mucin with altered properties (or stability) leading to an increased propensity for disease. In this work the enzymatic and biological properties of the mutant and wild type transferases will be characterized in order to understand their biological functions (i.e. role in MUC2 glycosylation) and subsequent role in disease. It is anticipated that these studies will lead to potential modalities for the detection, prevention, and/or treatment of colon cancer and other diseases.
In Aim 1 ppGalNAc T peptide and glycopeptide substrate specificities will be characterized for a series of wild type ppGalNAc Ts that have been implicated in human disease or have significantly interesting biochemical or biological properties.
In Aim 2 the mutant ppGalNAc T's, genetically linked to colon cancer, will be further characterized with respect to their enzymology, substrate specificities (including MUC2) and general cell biology (i.e. subcellular location and secretion). The goals of this aim will be to ultimately understand the association of these mutant transferases with MUC2 glycosylation and colon cancer. This work will also provide the first detailed biochemical structure-function analysis of this class of transferases.
In Aim 3 studies are extended to characterize the glycopeptide substrate preferences of transferases that transfer subsequent sugars to the peptide linked GalNAc i.e. (2-Gal (1-3), 2-GlcNAc (1-3) &1-NeuNAc (2-6)), initializing the first elongation steps of O-glycan biosynthesis. Alterations in these transferases (i.e. the Core 1, Core 3, and ST6GalNAc I &II transferases, respectively) also have associations with disease and various cancers. As a result of this work a basic understanding of the role of peptide sequence and local environment on the modulation of the initial steps of O-glycan elongation will be obtained. This work will contribute to the elucidation of the biological role(s) of these transferases and their impact on disease. Tools for the rational prediction of site specific O-glycosylation will also result from these studies.

Public Health Relevance

Mutations in a series of glycosyltransferases (protein glycosylating enzymes) have recently been found in patients with colon cancer. In this work we will study the enzymatic and biological properties of a series of mutant and wild type transferases to understand their biological functions and roles in this disease. It is anticipated that these studies will lead to potential modalities for the detection, prevention, and/or treatment of colon cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA078834-14
Application #
8271339
Study Section
Intercellular Interactions (ICI)
Program Officer
Sathyamoorthy, Neeraja
Project Start
1999-04-05
Project End
2013-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
14
Fiscal Year
2012
Total Cost
$267,715
Indirect Cost
$97,196
Name
Case Western Reserve University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Haugstad, Kristin E; Hadjialirezaei, Soosan; Stokke, Bjørn T et al. (2016) Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions. Glycobiology 26:1338-1350
Revoredo, Leslie; Wang, Shengjun; Bennett, Eric Paul et al. (2016) Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family. Glycobiology 26:360-76
Venkitachalam, Srividya; Revoredo, Leslie; Varadan, Vinay et al. (2016) Biochemical and functional characterization of glycosylation-associated mutational landscapes in colon cancer. Sci Rep 6:23642
Haugstad, Kristin E; Stokke, Bjørn T; Brewer, C Fred et al. (2015) Single molecule study of heterotypic interactions between mucins possessing the Tn cancer antigen. Glycobiology 25:524-34
Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram et al. (2015) Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis. Glycobiology 25:55-65
Boltin, Doron; Niv, Yaron (2013) Mucins in Gastric Cancer - An Update. J Gastrointest Dig Syst 3:15519
Gerken, Thomas A; Revoredo, Leslie; Thome, Joseph J C et al. (2013) The lectin domain of the polypeptide GalNAc transferase family of glycosyltransferases (ppGalNAc Ts) acts as a switch directing glycopeptide substrate glycosylation in an N- or C-terminal direction, further controlling mucin type O-glycosylation. J Biol Chem 288:19900-14
Haugstad, Kristin E; Gerken, Thomas A; Stokke, Bjørn T et al. (2012) Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level. Biomacromolecules 13:1400-9
Raman, Jayalakshmi; Guan, Yu; Perrine, Cynthia L et al. (2012) UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases: completion of the family tree. Glycobiology 22:768-77
Gerken, Thomas A (2012) O-glycoprotein biosynthesis: site localization by Edman degradation and site prediction based on random peptide substrates. Methods Mol Biol 842:81-108

Showing the most recent 10 out of 29 publications