Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols.
Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 371-376). Increased polyamine uptake correlates with an increased number of positively charged N-unsubstituted glucosamine units in the otherwise polyanionic heparan sulfate chains of glypican-1. During intracellular recycling of glypican-1, there is an NO-dependent deaminative cleavage of heparan sulfate at these glucosamine units, which would eliminate the positive charges (Ding, K., Sandgren, S., Mani, K., Belting, M., and Fransson, L.-A. (2001) J. Biol. Chem. 276, 46779-46791). Here, using both biochemical and microscopic techniques, we have identified and isolated S-nitrosylated forms of glypican-1 as well as slightly charged glypican-1 glycoforms containing heparan sulfate chains rich in N-unsubstituted glucosamines. These glycoforms were converted to highly charged species upon treatment of cells with 1 mm l-ascorbate, which releases NO from nitrosothiols, resulting in deaminative cleavage of heparan sulfate at the N-unsubstituted glucosamines. S-Nitrosylation and subsequent deaminative cleavage were abrogated by inhibition of a Cu(2+)/Cu(+) redox cycle. Under cell-free conditions, purified S-nitrosylated glypican-1 was able to autocleave its heparan sulfate chains when NO release was triggered by l-ascorbate. The heparan sulfate fragments generated in cells during this autocatalytic process contained terminal anhydromannose residues. We conclude that the core protein of glypican-1 can slowly accumulate NO as nitrosothiols, whereas Cu(2+) is reduced to Cu(+). Subsequent release of NO results in efficient deaminative cleavage of the heparan sulfate chains attached to the same core protein, whereas Cu(+) is oxidized to Cu(2+).
Ding
Kan
K
Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden.
Mani
Katrin
K
Cheng
Fang
F
Belting
Mattias
M
Fransson
Lars-Ake
LA
D002384:Catalysis; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007477:Ions; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D026403:S-Nitrosothiols / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D019812:Heparan Sulfate Proteoglycans; D007477:Ions; D020033:Protein Isoforms; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D003300:Copper