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Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
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Proteolytic Cleavage Driven by Glycosylation.

Miriam P Kötzler1, Stephen G Withers2

  • 1From the Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.

The Journal of Biological Chemistry
|October 31, 2015
PubMed
Summary

Proteolytic processing of human host cell factor 1 (HCF-1) involves a novel mechanism. This study provides evidence that a covalent glycosyl ester intermediate directly leads to the proteolysis of HCF-1, supporting a proposed enzymatic pathway.

Keywords:
glutamateglutamyl esterglycobiologyglycosidaseglycosyl esterglycosylationprotein processingprotein splicingproteolysispyroglutamate

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Human host cell factor 1 (HCF-1) undergoes post-translational modification.
  • O-GlcNAc-transferase (OGT) was unexpectedly found to mediate HCF-1 processing.
  • A mechanism involving a covalent glycosyl ester intermediate was proposed but lacked direct evidence.

Purpose of the Study:

  • To investigate the proposed mechanism of HCF-1 proteolytic processing.
  • To provide experimental evidence for the formation and role of a glycosyl ester intermediate.
  • To support the proposed UDP-GlcNAc-dependent pathway mediated by OGT.

Main Methods:

  • Utilized a model enzyme system with established glycosyl ester formation.
  • Analyzed the consequences of glycosyl ester formation on adjacent peptide bonds.
  • Investigated the potential for intramolecular rearrangement leading to proteolysis.

Main Results:

  • Demonstrated that glycosyl ester formation within an enzyme active site can induce proteolysis.
  • Provided substantive support for the proposed mechanism of HCF-1 processing.
  • Confirmed the potential for a covalent glycosyl ester adduct to undergo rearrangement and cleave a peptide bond.

Conclusions:

  • The formation of a covalent glycosyl ester intermediate is a viable mechanism for HCF-1 proteolysis.
  • This study validates the proposed pathway for OGT-mediated HCF-1 processing.
  • The findings offer significant insight into the enzymatic regulation of HCF-1 maturation.