A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells.
We show here that the endothelial cell-line ECV 304 expresses the heparan sulfate proteoglycan glypican-1. The predominant cellular glycoform carries truncated side-chains and is accompanied by heparan sulfate oligosaccharides. Treatment with brefeldin A results in accumulation of a glypican proteoglycan with full-size side-chains while the oligosaccharides disappear. During chase the glypican proteoglycan is converted to partially degraded heparan sulfate chains and chain-truncated proteoglycan, both of which can be captured by treatment with suramin. The heparan sulfate chains in the intact proteoglycan can be depolymerized by nitrite-dependent cleavage at internally located N-unsubstituted glucosamine moieties. Inhibition of NO-synthase or nitrite-deprivation prevents regeneration of intact proteoglycan from truncated precursors as well as formation of oligosaccharides. In nitrite-deprived cells, formation of glypican proteoglycan is restored when NO-donor is supplied. We propose that, in recycling glypican-1, heparan sulfate chains are cleaved at or near glucosamines with unsubstituted amino groups. NO-derived nitrite is then required for the removal of short, nonreducing terminal saccharides containing these N-unsubstituted glucosamine residues from the core protein stubs, facilitating re-synthesis of heparan sulfate chains.
Mani
K
K
Department of Cell and Molecular Biology, Lund University, Sweden.
Jönsson
M
M
Edgren
G
G
Belting
M
M
Fransson
L A
LA
D020126:Brefeldin A / Q000494:pharmacology; D002461:Cell Line, Transformed; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009573:Nitrites / Q000037:antagonists & inhibitors / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology
D020126:Brefeldin A; D004791:Enzyme Inhibitors; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009573:Nitrites; D009844:Oligosaccharides; D011500:Protein Synthesis Inhibitors; D013498:Suramin