The Structural Role of N-Linked Glycans on Human Glypican-1
Svensson Birkedal, Gabriel LU ; Wittsten, Axel Hyrenius ; Linse, Sara LU and Mani, Katrin LU
(2011)
In Biochemistry
50(43).
p.9377-9387
- Abstract
- Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was... (More)
- Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79QN116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/2208063
- author
- Svensson Birkedal, Gabriel
LU
; Wittsten, Axel Hyrenius
; Linse, Sara
LU
and Mani, Katrin
LU
- organization
- publishing date
- 2011
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biochemistry
- volume
- 50
- issue
- 43
- pages
- 9377 - 9387
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- wos:000296217000020
- scopus:80055018082
- pmid:21932778
- ISSN
- 0006-2960
- DOI
- 10.1021/bi200218s
- language
- English
- LU publication?
- yes
- id
- a254b77e-df3b-4b04-9eb6-f8d66a0c321c (old id 2208063)
- date added to LUP
- 2016-04-01 10:36:59
- date last changed
- 2023-09-28 09:53:24
@article{a254b77e-df3b-4b04-9eb6-f8d66a0c321c,
abstract = {{Glypicans are cell-surface heparan sulfate proteoglycans that regulate developmental signaling pathways by binding growth factors to their heparan sulfate chains. The primary structures of glypican core proteins contain potential N-glycosylation sites, but the importance of N-glycosylation in glypicans has never been investigated in detail. Here, we studied the role of the possible N-glycosylation sites at Asn-79 and Asn-116 in recombinant anchorless glypican-1 expressed in eukaryotic cells. Mutagenesis and enzymatic cleavage indicated that the potential N-glycosylation sites are invariably occupied. Experiments using the drug tunicamycin to inhibit the N-linked glycosylation of glypican-1 showed that secretion of anchorless glypican-1 was reduced and that the protein did not accumulate inside the cells. Heparan sulfate substitution of N-glycosylation mutant N116Q was similar to wild-type glypican-1 while the N79Q mutant and also the double mutant N79QN116Q were mostly secreted as high-molecular-weight heparan sulfate proteoglycan. N-Glycosylation mutants and N-deglycosylated glypican-1 had far-UV circular dichroism and fluorescence emission spectra that were highly similar to those of N-glycosylated glypican-1. A single unfolding transition at high concentrations of urea was found for both N-deglycosylated glypican-1 and glypican-1 in which the N-glycosylation sites had been removed by mutagenesis when chemical denaturation was monitored by circular dichroism and fluorescence emission spectroscopy. In summary, we have found that the potential N-glycosylation sites in glypican-1 are invariably occupied and that the N-linked glycans on glypican-1 affect protein expression and heparan sulfate substitution but that correct folding can be obtained in the absence of N-linked glycans.}},
author = {{Svensson Birkedal, Gabriel and Wittsten, Axel Hyrenius and Linse, Sara and Mani, Katrin}},
issn = {{0006-2960}},
language = {{eng}},
number = {{43}},
pages = {{9377--9387}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Biochemistry}},
title = {{The Structural Role of N-Linked Glycans on Human Glypican-1}},
url = {{http://dx.doi.org/10.1021/bi200218s}},
doi = {{10.1021/bi200218s}},
volume = {{50}},
year = {{2011}},
}