Involvement of Heparan Sulphate Biosynthesis and Turnover in Cell Proliferation. A Novel Role for Nitric Oxide in Recycling of Heparan Sulphate Proteoglycans

Abstract: The present investigation focuses on the role of HS metabolism in cell proliferation. The effect of the HS priming ß-D-xyloside, 2-(6-hydroxynaphthyl)-O-ß-D-xylopyranoside (Xyl-2-Nap-6-OH) on the proliferation of normal and transformed cells was studied. Xyl-2-Nap-6-OH inhibited growth of transformed cells to a significantly greater extent than normal cells. The growth inhibition exerted by the xyloside was believed to be due to its ability to prime GAG synthesis since the non-priming L-isomer did not possess any antiproliferative activity. The molecular structure of the aglycone and its ability to prime HS were other features assumed to be of importance for its antiproliferative activity. Recycling of HSPG may be a vehicle for endo/exocytosis of HS-binding growth factors and polyamines and thereby regulating cell proliferation. HSPG metabolism/recycling was therefore extensively studied in transformed endothelial cells. The major focus was on the role of NO-derived nitrite in the turnover of HSPG. As N-unsubstituted GlcN in HS chains are potential sites for NO-generated cleavage, the content and location of such residues was investigated. Transformed endothelial cells expressed a HSPG with a core protein of 60-70 kDa, which was recognised by a polyclonal antiserum raised against recombinant glypican-1 protein. In unperturbed cells, most of the radiolabelled glypican-1 carried truncated HS chains accompanied by HS oligosaccharides. Treatment with brefeldin A, which inhibits transport from the ER to the Golgi and also exit from the TGN and/or endocytosis in polarised cells, resulted in accumulation of glypican PG with full-size side chains while oligosaccharides disappeared. Treatment with suramin, an inhibitor of endoheparanase, led to partial inhibition of degradation of HS. Glypican-1 glycoforms in brefeldin A-treated cells contained long HS chains with GlcNH2 residues in multiple places, whereas unperturbed cells, suramin-treated cells, and nitrite-deprived cells contained short HS chains with only a few GlcNH2 residues. The number of GlcNH2 residues increased by combined suramin treatment and nitrite deprivation. This suggested that GlcNH2 residues and endoheparanase cleavage sites were closely located in HS chains. Pulse-chase studies clearly indicated that suramin arrested chains were the precursor of large-size PGs in the absence of de novo glypican-1 core protein synthesis. Recycling of suramin-arrested chains back to brefeldin A-arrested large PGs was precluded by nitrite deprivation. Formation of brefeldin A-arrested large glypican PGs was restored when NO-donor was supplied to nitrite-deprived cells. Taken together our data suggest a recycling of glypican-1 in transformed endothelial cells. During recycling, there is endoglycosidic cleavage of HS at or near GlcNH2 residues, and removal of these short nonreducing terminal GlcNH2 containing saccharides by NO derived nitrite would provide fresh acceptor-sites for HS chain extension. In order to assess the relationship between HS priming and antiproliferative activity of naphthol-containing ß-D-xylosides, cell proliferation assays and GAG priming studies were performed in the presence of nitrite depriving drugs. Different xylosides were synthesised and tested for growth inhibition and priming of HS synthesis. The selective growth-inhibitory effect of Xyl-2-Nap-6-OH appeared to be quite specific for this compound. Xylosides without the 6-hydroxyl, with an O-methylated 6-hydroxyl, with a free hydroxyl in a different position, or with non-fused aromatic rings were not antiproliferative. All of the tested naphthol-based xylosides were capable of priming HS synthesis. Inhibition of degradation by suramin or nitrite-deprivation resulted in increased intracellular accumulation of HS chains. Interestingly, nitrite-deprivation abrogated the growth inhibitory effect of Xyl-2-Nap-6-OH. We propose that Xyl-2-Nap-6-OH initiates synthesis of HS chains with occasional N-unsubstituted GlcN residues and that these chains can be taken up by the cells and degraded to bioactive compounds in the presence of nitrite.