Journal of Biological Chemistry
Volume 278, Issue 47, 21 November 2003, Pages 47181-47189
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Glycobiology and Extracellular Matrices
Glypican-1 Is a Vehicle for Polyamine Uptake in Mammalian Cells: A PIVOTAL ROLE FOR NITROSOTHIOL-DERIVED NITRIC OXIDE*

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Polyamines (putrescine, spermidine, and spermine) are essential for growth and survival of all cells. When polyamine biosynthesis is inhibited, there is up-regulation of import. The mammalian polyamine transport system is unknown. We have previously shown that the heparan sulfate (HS) side chains of recycling glypican-1 (Gpc-1) can sequester spermine, that intracellular polyamine depletion increases the number of NO-sensitive N-unsubstituted glucosamines in HS, and that NO-dependent cleavage of HS at these sites is required for spermine uptake. The NO is derived from S-nitroso groups in the Gpc-1 protein. Using RNA interference technology as well as biochemical and microscopic techniques applied to both normal and uptake-deficient cells, we demonstrate that inhibition of Gpc-1 expression abrogates spermine uptake and intracellular delivery. In unperturbed cells, spermine and recycling Gpc-1 carrying HS chains rich in N-unsubstituted glucosamines were co-localized. By exposing cells to ascorbate, we induced release of NO from the S-nitroso groups, resulting in HS degradation and unloading of the sequestered polyamines as well as nuclear targeting of the deglycanated Gpc-1 protein. Polyamine uptake-deficient cells appear to have a defect in the NO release mechanism. We have managed to restore spermine uptake partially in these cells by providing spermine NONOate and ascorbate. The former bound to the HS chains of recycling Gpc-1 and S-nitrosylated the core protein. Ascorbate released NO, which degraded HS and liberated the bound spermine. Recycling HS proteoglycans of the glypican-type may be plasma membrane carriers for cargo taken up by caveolar endocytosis.

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*

This work was supported by Swedish Science Council Grants VR-M and VR-NaTe and by grants from the Cancer Fund; the Strategic Research Fund (Glycoconjugates in Biological Systems) (to F. C.); Polysackaridforskning i Uppsala AB; the WennerGren, Tegger, Kock, and Österlund Foundations; Xylogen AB; and the Medical Faculty of Lund University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Both authors contributed equally to this work.

Present address: Depts. of Immunology and Vascular Biology, Scripps Research Inst., La Jolla, CA 92014.

Present address: Dept. of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, CA 92697-2300.