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The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.

Mani, Katrin LU ; Cheng, Fang LU ; Sandgren, Staffan LU ; Van Den Born, Jacob; Havsmark, Birgitta LU ; Ding, Kan and Fransson, Lars-Åke LU (2004) In Glycobiology 14(7). p.599-607
Abstract
The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide–generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly... (More)
The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide–generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide–depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
heparanase, heparan sulfate, nitric oxide, mAb 10E4, glypican-1
in
Glycobiology
volume
14
issue
7
pages
599 - 607
publisher
Oxford University Press
external identifiers
  • pmid:15044385
  • wos:000222034500004
  • scopus:3142664810
ISSN
1460-2423
DOI
10.1093/glycob/cwh067
language
English
LU publication?
yes
id
afa188a2-6869-4633-aaae-bda0eb65b020 (old id 120998)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15044385&dopt=Abstract
date added to LUP
2007-06-26 11:13:45
date last changed
2017-01-01 06:40:48
@article{afa188a2-6869-4633-aaae-bda0eb65b020,
  abstract     = {The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide–generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide–depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.},
  author       = {Mani, Katrin and Cheng, Fang and Sandgren, Staffan and Van Den Born, Jacob and Havsmark, Birgitta and Ding, Kan and Fransson, Lars-Åke},
  issn         = {1460-2423},
  keyword      = {heparanase,heparan sulfate,nitric oxide,mAb 10E4,glypican-1},
  language     = {eng},
  number       = {7},
  pages        = {599--607},
  publisher    = {Oxford University Press},
  series       = {Glycobiology},
  title        = {The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.},
  url          = {http://dx.doi.org/10.1093/glycob/cwh067},
  volume       = {14},
  year         = {2004},
}