Advanced

S-Nitrosylation of secreted recombinant human glypican-1.

Svensson Birkedal, Gabriel LU and Mani, Katrin LU (2009) In Glycoconjugate Journal 26. p.1247-1257
Abstract
Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to... (More)
Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Glycoconjugate Journal
volume
26
pages
1247 - 1257
publisher
Springer
external identifiers
  • wos:000272784000013
  • pmid:19479373
  • scopus:77649252981
ISSN
1573-4986
DOI
10.1007/s10719-009-9243-z
language
English
LU publication?
yes
id
adbf4e3c-546d-45fa-8e7f-5029193b5f4d (old id 1411872)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/19479373?dopt=Abstract
date added to LUP
2009-06-03 15:08:31
date last changed
2017-04-23 04:38:21
@article{adbf4e3c-546d-45fa-8e7f-5029193b5f4d,
  abstract     = {Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.},
  author       = {Svensson Birkedal, Gabriel and Mani, Katrin},
  issn         = {1573-4986},
  language     = {eng},
  pages        = {1247--1257},
  publisher    = {Springer},
  series       = {Glycoconjugate Journal},
  title        = {S-Nitrosylation of secreted recombinant human glypican-1.},
  url          = {http://dx.doi.org/10.1007/s10719-009-9243-z},
  volume       = {26},
  year         = {2009},
}