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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1507124
- author
- Svensson Birkedal, Gabriel
LU
; Linse, Sara
LU
and Mani, Katrin
LU
- organization
- publishing date
- 2009
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemistry
- volume
- 48
- issue
- 42
- pages
- 9994 - 10004
- publisher
- The American Chemical Society (ACS)
- 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
- 2016-04-01 12:34:07
- date last changed
- 2023-09-16 12:38:40
@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 (ACS)}},
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}},
doi = {{10.1021/bi901402x}},
volume = {{48}},
year = {{2009}},
}