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Characterization and Crystallization of Anchorless Glypican-1

Svensson Birkedal, Gabriel LU (2011) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2011:5.
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.... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Couchman, John Robert, Department of Biomedical Sciences, University of Copenhagen
organization
publishing date
type
Thesis
publication status
published
subject
keywords
S-nitrosylation, nitric oxide, protein stability, protein conformation, X-ray crystallography, calorimetry, spectroscopy, proteoglycan, glypican, heparan sulfate
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2011:5
pages
125 pages
publisher
Department of Experimental Medical Science, Lund Univeristy
defense location
Segerfalk Lecture Hall, BMC Neuroscience Centre
defense date
2011-01-21 13:00
ISSN
1652-8220
ISBN
978-91-86671-51-8
language
English
LU publication?
yes
id
1115c9fb-08d2-4c6b-8794-74f36dadbcce (old id 1747500)
date added to LUP
2010-12-22 07:57:53
date last changed
2016-09-19 08:44:48
@phdthesis{1115c9fb-08d2-4c6b-8794-74f36dadbcce,
  abstract     = {Glypicans are cell-surface heparan sulfate proteoglycans that regulate Wnt,<br/><br>
Hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling<br/><br>
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.<br/><br>
In this work, we studied anchorless glypican-1 expressed in mammalian<br/><br>
cells. The folding and conformational stability of anchorless glypican-1 was<br/><br>
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.<br/><br>
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<br/><br>
method and diffracted to 2.8 Å resolution using synchrotron radiation.},
  author       = {Svensson Birkedal, Gabriel},
  isbn         = {978-91-86671-51-8},
  issn         = {1652-8220},
  keyword      = {S-nitrosylation,nitric oxide,protein stability,protein conformation,X-ray crystallography,calorimetry,spectroscopy,proteoglycan,glypican,heparan sulfate},
  language     = {eng},
  pages        = {125},
  publisher    = {Department of Experimental Medical Science, Lund Univeristy},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Characterization and Crystallization of Anchorless Glypican-1},
  volume       = {2011:5},
  year         = {2011},
}