Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivital role for nitrosothiol-derived nitric oxide.
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.
Belting
Mattias
M
Department of Cell and Molecular Biology, Lund University, SE-221 84 Lund, Sweden.
Mani
Katrin
K
Jönsson
Mats
M
Cheng
Fang
F
Sandgren
Staffan
S
Jonsson
Susanne
S
Ding
Kan
K
Delcros
Jean-Guy
JG
Fransson
Lars-Ake
LA
D001205:Ascorbic Acid / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009603:Nitroso Compounds / Q000378:metabolism; D011073:Polyamines / Q000378:metabolism / Q000493:pharmacokinetics*; D013096:Spermine / Q000493:pharmacokinetics; D014162:Transfection
D001205:Ascorbic Acid; D002352:Carrier Proteins; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D009603:Nitroso Compounds; D011073:Polyamines; D013096:Spermine; C496314:NOS3 protein, human