Characterization and Crystallization of Anchorless Glypican-1

Gabriel Svensson Birkedal

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Glypicans are cell-surface heparan sulfate proteoglycans that regulate Wnt,
Hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling
through their heparan sulfate chains. Recent studies have shown that glypican core proteins also have functional roles in growth factor signaling, but biochemical and structural knowledge regarding the core proteins is limited. Glypican-1 is involved in brain development and is one of six members of the mammalian family of glypicans.
In this work, we studied anchorless glypican-1 expressed in mammalian
cells. The folding and conformational stability of anchorless glypican-1 was
investigated using spectroscopic and calorimetric techniques. Further, nitric oxide modification of cysteine residues was investigated using a biotin labeling method. We also investigated the role of N-glycosylation on protein folding, secretion, and heparan sulfate substitution. Moreover, anchorless glypican-1 was subjected to high-throughput crystallization screening.
The results showed that glypican-1 is a stable α-helical protein and that the proteoglycan form is protected from heat-induced aggregation. In addition, it was found that nitric oxide can be covalently attached to cysteine residues in anchorless glypican-1, forming S-nitrosothiols. Furthermore, the potential N-glycosylation sites in glypican-1 were found to be invariably occupied and the N-linked glycans of glypican-1 were found to affect protein expression and heparan sulfate substitution, but correct folding could be obtained in the absence of N-linked glycans. Finally, crystals of purified glypican-1 were obtained by vapordiffusion
method and diffracted to 2.8 Å resolution using synchrotron radiation.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Glycobiology
Supervisors/Advisors
  • Mani, Katrin, Supervisor
  • Fransson, Lars-Åke, Supervisor
Award date2011 Jan 21
Publisher
Print ISBNs978-91-86671-51-8
Publication statusPublished - 2011

Bibliographical note

Defence details

Date: 2011-01-21
Time: 13:00
Place: Segerfalk Lecture Hall, BMC Neuroscience Centre

External reviewer(s)

Name: Couchman, John Robert
Title: Professor
Affiliation: Department of Biomedical Sciences, University of Copenhagen

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Subject classification (UKÄ)

  • Medicinal Chemistry

Keywords

  • S-nitrosylation
  • nitric oxide
  • protein stability
  • protein conformation
  • X-ray crystallography
  • calorimetry
  • spectroscopy
  • proteoglycan
  • glypican
  • heparan sulfate

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