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Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation

Perrine, Cynthia L.
http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997

Abstract Details

2009, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Many secreted and membrane-associated proteins of eukaryotic cells contain heavily O-glycosylated mucin domains. Glycoproteins which contain such domains play key biological functions, i.e., cell-cell adhesion, host pathogen interaction, and intracellular protein trafficking. Mucin type O-glycosylation occurs exclusively in the Golgi complex beginning with the transfer of GalNAc from UDP-GalNAc to a serine or threonine residue in the polypeptide catalyzed by a large family of polypeptide N-α-acetyl galactosaminyltransferases (ppGalNAcTs). The α-GalNAc-O-Ser/Thr product forms the core carbohydrate structure which is elongated in the Golgi by specific transferases. The peptide substrate specificities of these transferases are largely unknown. Based on earlier random peptide studies, I have determined the glycopeptide substrate specificities of the catalytic domains of ppGalNAc T1, T2, and T10 utilizing two complementary random glycopeptides, UDP-[3H]-GalNAc, and the azido-labeled UDP-GalNAc analogue, UDP-GalNAz. ppGalNAc T10 was shown to prefer Ser or Thr-O-GalNAc at the +1 position (relative to the site of glycosylation) with a large ~10 fold enhancement. ppGalNAc T1 and T2 gave little or no Ser/Thr-O-GalNAc preferences as expected from previous studies on these transferases. Peptide sequence and neighboring glycosylation effects on the specificity of T-synthase, which transfers β Gal to the 3 position of GalNAc, was also determined using a random glycopeptide substrate. Novel enhancements for Phe and Tyr at the +3 and +4 positions relative to the site of glycosylation were observed with a smaller enhancement for Gly at the +1 position. An overall preference for negatively charged amino acids Glu and Asp was also observed. Neighboring glycosylation was also examined for T-synthase by glycosylating the porcine mucin tandem repeat. A candidate ppGalNAc T1 inhibitor peptide was also synthesized based on previous studies by our laboratory. The inhibitor peptide contained a photocross-linking p-nitro-phenylalanine derivative. The inhibitor peptide moderately decreased activity in the enzymes, but showed no specificity against ppGalNAc T1 or T2. As a result of these studies, robust methods for obtaining and quantifying the peptide and glycopeptide specificities of the individual transferases that initiate mucin O-glycosylation are now available. The development of methods for predicting transferase specific glycosylation are now possible based on this work.
Thomas Gerken (Advisor)
Clemens Burda (Committee Chair)
John Stuehr (Committee Member)
Thomas Kelley (Committee Member)
251 p.
Glycosylation; Glycoproteins; Mucins; UDP-GalNAc; ppGalNAcTs

Recommended Citations

Citations

  • Perrine, C. L. (2009). Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997

    APA Style (7th edition)

  • Perrine, Cynthia. Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation. 2009. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997.

    MLA Style (8th edition)

  • Perrine, Cynthia. "Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation." Doctoral dissertation, Case Western Reserve University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997

    Chicago Manual of Style (17th edition)

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© 2009, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.