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Chemical and Thermal Unfolding of Glypican-1: Protective Effect of Heparan Sulfate against Heat-Induced Irreversible Aggregation

Svensson Birkedal, Gabriel LU ; Linse, Sara LU and Mani, Katrin LU (2009) In Biochemistry 48(42). p.9994-10004
Abstract
Glypicans are cell-surface heparan sulfate proteoglycans that influence Wnt, hedgehog, decapentaplegic, and fibroblast growth factor activity via their heparan sulfate chains. However, recent studies have shown that glypican core proteins also have a role in growth factor signaling. Here, we expressed secreted recombinant human glypican-1 in eukaryotic cells. Recombinant glypican-1 was expressed as two glycoforms, one as proteoglycan substituted with heparan sulfate chains and one as the core protein devoid of glycosaminoglycans. Far-UV circular dichroism (CD) analysis of glypican-1 isolated under native conditions showed that the glypican-1 core protein is predominantly alpha-helical in structure, with identical spectra for the core... (More)
Glypicans are cell-surface heparan sulfate proteoglycans that influence Wnt, hedgehog, decapentaplegic, and fibroblast growth factor activity via their heparan sulfate chains. However, recent studies have shown that glypican core proteins also have a role in growth factor signaling. Here, we expressed secreted recombinant human glypican-1 in eukaryotic cells. Recombinant glypican-1 was expressed as two glycoforms, one as proteoglycan substituted with heparan sulfate chains and one as the core protein devoid of glycosaminoglycans. Far-UV circular dichroism (CD) analysis of glypican-1 isolated under native conditions showed that the glypican-1 core protein is predominantly alpha-helical in structure, with identical spectra for the core protein and the proteoglycan form. The conformational stability of glypican-1 core protein to urea and guanidine hydrochloride denaturation was monitored by CD and fluorescence spectroscopy and showed a single unfolding transition at high concentrations of the denaturant (5.8 and 2.6 M, respectively). Renaturation from guanidine hydrochloride gave far-UV CD and fluorescence spectra identical to the spectra of native glypican-1. Thermal denaturation monitored by CD and differential scanning calorimetry (DSC) showed a single structural transition at a temperature of similar to 70 degrees C. Refolding of the heat-denatured glypican-1 core protein was dependent on protein concentration, suggesting that intermolecular interactions are involved in irreversible denaturation. However, refolding was concentration-independent for the proteoglycan form, suggesting that O-glycosylation protects the protein from irreversible aggregation. In summary, we have shown that the glypican-1 core protein is a stable CL-helical protein and that the proteoglycan form of glypican-1 is protected from heat-induced aggregation. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
48
issue
42
pages
9994 - 10004
publisher
The American Chemical Society
external identifiers
  • wos:000270810000006
  • scopus:70350218956
ISSN
0006-2960
DOI
10.1021/bi901402x
language
English
LU publication?
yes
id
36d741f6-42d3-47e2-993a-29b661cd1ec5 (old id 1507124)
date added to LUP
2009-11-23 10:23:15
date last changed
2017-01-01 05:15:34
@article{36d741f6-42d3-47e2-993a-29b661cd1ec5,
  abstract     = {Glypicans are cell-surface heparan sulfate proteoglycans that influence Wnt, hedgehog, decapentaplegic, and fibroblast growth factor activity via their heparan sulfate chains. However, recent studies have shown that glypican core proteins also have a role in growth factor signaling. Here, we expressed secreted recombinant human glypican-1 in eukaryotic cells. Recombinant glypican-1 was expressed as two glycoforms, one as proteoglycan substituted with heparan sulfate chains and one as the core protein devoid of glycosaminoglycans. Far-UV circular dichroism (CD) analysis of glypican-1 isolated under native conditions showed that the glypican-1 core protein is predominantly alpha-helical in structure, with identical spectra for the core protein and the proteoglycan form. The conformational stability of glypican-1 core protein to urea and guanidine hydrochloride denaturation was monitored by CD and fluorescence spectroscopy and showed a single unfolding transition at high concentrations of the denaturant (5.8 and 2.6 M, respectively). Renaturation from guanidine hydrochloride gave far-UV CD and fluorescence spectra identical to the spectra of native glypican-1. Thermal denaturation monitored by CD and differential scanning calorimetry (DSC) showed a single structural transition at a temperature of similar to 70 degrees C. Refolding of the heat-denatured glypican-1 core protein was dependent on protein concentration, suggesting that intermolecular interactions are involved in irreversible denaturation. However, refolding was concentration-independent for the proteoglycan form, suggesting that O-glycosylation protects the protein from irreversible aggregation. In summary, we have shown that the glypican-1 core protein is a stable CL-helical protein and that the proteoglycan form of glypican-1 is protected from heat-induced aggregation.},
  author       = {Svensson Birkedal, Gabriel and Linse, Sara and Mani, Katrin},
  issn         = {0006-2960},
  language     = {eng},
  number       = {42},
  pages        = {9994--10004},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Chemical and Thermal Unfolding of Glypican-1: Protective Effect of Heparan Sulfate against Heat-Induced Irreversible Aggregation},
  url          = {http://dx.doi.org/10.1021/bi901402x},
  volume       = {48},
  year         = {2009},
}