The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.
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.
Mani
Katrin
K
Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden.
Cheng
Fang
F
Sandgren
Staffan
S
Van Den Born
Jacob
J
Havsmark
Birgitta
B
Ding
Kan
K
Fransson
Lars-Ake
LA
D000911:Antibodies, Monoclonal; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005944:Glucosamine / Q000276:immunology / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019812:Heparan Sulfate Proteoglycans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D009569:Nitric Oxide; D013498:Suramin; D019001:Nitric Oxide Synthase; D005944:Glucosamine