Nitric oxide-dependent processing of heparan sulfate in recycling S-nitrosylated glypican-1 takes place in caveolin-1-containing endosomes.
We have previously demonstrated intracellular degradation of the heparan sulfate side chains in recycling glypican-1 by heparanase and by deaminative cleavage at N-unsubstituted glucosamine with nitric oxide derived from intrinsic nitrosothiols (see Ding, K., Mani, K., Cheng, F., Belting, M. and Fransson, L.-A. (2002) J. Biol. Chem. 277, 33353-33360). To determine where and in what order events take place, we have visualized, by using confocal laser-scanning immunofluorescence microscopy, glypican-1 variants in unperturbed cells or arrested at various stages of processing. In unperturbed proliferating cells, glypican-1 was partly S-nitrosylated. Intracellular glypican-1 was enriched in endosomes, colocalized significantly with GM-1 ganglioside, caveolin-1, and Rab9-positive endosomes, and carried side chains rich in N-unsubstituted glucosamine residues. However, such residues were scarce in cell surface glypican-1. Brefeldin A-arrested glypican-1, which was non-S-nitrosylated and carried side chains rich in N-unsubstituted glucosamines, colocalized extensively with caveolin-1 but not with Rab9. Suramin, which inhibits heparanase, induced the appearance of S-nitrosylated glypican-1 in caveolin-1-rich compartments. Inhibition of deaminative cleavage did not prevent heparanase from generating heparan sulfate oligosaccharides that colocalized strongly with caveolin-1. Growth-quiescent cells displayed extensive NO-dependent deaminative cleavage of heparan sulfate-generating anhydromannose-terminating fragments that were partly associated with acidic vesicles. Proliferating cells generated such fragments during polyamine uptake. We conclude that recycling glypican-1 that is associated with caveolin-1-containing endosomes undergoes sequential N-desulfation/N-deacetylation, heparanase cleavage, S-nitrosylation, NO release, and deaminative cleavage of its side chains in conjunction with polyamine uptake.
Cheng
Fang
F
Department of Cell and Molecular Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden and the Department of Cell Biology, Free University of Amsterdam, Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands.
Mani
Katrin
K
van den Born
Jacob
J
Ding
Kan
K
Belting
Mattias
M
Fransson
Lars-Ake
LA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism*; D005944:Glucosamine / Q000737:chemistry; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism; D013498:Suramin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000911:Antibodies, Monoclonal; D020126:Brefeldin A; D051242:Caveolin 1; D022461:Caveolins; D005944:Glucosamine; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D009569:Nitric Oxide; D009844:Oligosaccharides; D011073:Polyamines; D013498:Suramin; C495540:CAV1 protein, human