Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
Proteoglycan involvement in polyamine uptake.
We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki=0.16+/-0.04 microM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-beta-D-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by alpha-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or beta-D-xyloside, which might have future therapeutical implications. (+info)
To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'-phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)-binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, DeltaUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3(35)S+/-6S-++ +IdceA2S-[6-3H]Glc NS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HSact precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O-sulfate acceptor sites in HSact and HSinact precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences. (+info)
Frequent silencing of the GPC3 gene in ovarian cancer cell lines.
GPC3 encodes a glypican integral membrane protein and is mutated in the Simpson-Golabi-Behmel syndrome. Simpson-Golabi-Behmel syndrome, an X-linked condition, is characterized by pre- and postnatal overgrowth as well as by various other abnormalities, including increased risk of embryonal tumors. The GPC3 gene is located at Xq26, a region frequently deleted in advanced ovarian cancers. To determine whether GPC3 is a tumor suppressor in ovarian neoplasia, we studied its expression and mutational status in 13 ovarian cancer cell lines. No mutations were found in GPC3, but its expression was lost in four (31%) of the cell lines analyzed. In an of the cases where GPC3 expression was lost, the GPC3 promoter was hypermethylated, as demonstrated by Southern analysis. Expression of GPC3 was restored by treatment of the cells with the demethylating agent 5-aza-2'-deoxycytidine. A colony-forming assay confirmed that ectopic GPC3 expression inhibited the growth of ovarian cancer cell lines. Our results show that GPC3, a gene involved in the control of organ growth, is frequently inactivated in a subset of ovarian cancers and suggest that it may function as a tumor suppressor in the ovary. (+info)
Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through the receptor tyrosine kinase c-Met.
CD44 has been implicated in tumor progression and metastasis, but the mechanism(s) involved is as yet poorly understood. Recent studies have shown that CD44 isoforms containing the alternatively spliced exon v3 carry heparan sulfate side chains and are able to bind heparin-binding growth factors. In the present study, we have explored the possibility of a physical and functional interaction between CD44 and hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the receptor tyrosine kinase c-Met. The HGF/SF-c-Met pathway mediates cell growth and motility and has been implicated in tumor invasion and metastasis. We demonstrate that a CD44v3 splice variant efficiently binds HGF/SF via its heparan sulfate side chain. To address the functional relevance of this interaction, Namalwa Burkitt's lymphoma cells were stably co-transfected with c-Met and either CD44v3 or the isoform CD44s, which lacks heparan sulfate. We show that, as compared with CD44s, CD44v3 promotes: (i) HGF/SF-induced phosphorylation of c-Met, (ii) phosphorylation of several downstream proteins, and (iii) activation of the MAP kinases ERK1 and -2. By heparitinase treatment and the use of a mutant HGF/SF with greatly decreased affinity for heparan sulfate, we show that the enhancement of c-Met signal transduction induced by CD44v3 was critically dependent on heparan sulfate moieties. Our results identify heparan sulfate-modified CD44 (CD44-HS) as a functional co-receptor for HGF/SF which promotes signaling through the receptor tyrosine kinase c-Met, presumably by concentrating and presenting HGF/SF. As both CD44-HS and c-Met are overexpressed on several types of tumors, we propose that the observed functional collaboration might be instrumental in promoting tumor growth and metastasis. (+info)
Identification and characterization of ligands for L-selectin in the kidney. II. Expression of chondroitin sulfate and heparan sulfate proteoglycans reactive with L-selectin.
Ligands for the leukocyte adhesion molecule L-selectin are expressed not only in lymph node high endothelial venules (HEV) but also in the renal distal tubuli. Here we report that L-selectin-reactive molecules in the kidney are chondroitin sulfate and heparan sulfate proteoglycans of 500-1000 kDa, unlike those in HEV bearing sialyl Lewis X-like carbohydrates. Binding of L-selectin to these molecules was mediated by the lectin domain of L-selectin and required divalent cations. Binding was inhibited by chondroitinase and/or heparitinase but not sialidase. Thus, L-selectin can recognize chondroitin sulfate and heparan sulfate glycosaminoglycans structurally distinct from sialyl Lewis X-like carbohydrates. (+info)
Heparin and heparan sulphate protect basic fibroblast growth factor from non-enzymic glycosylation.
Non-enzymic glycosylation of basic fibroblast growth factor (bFGF, FGF-2) has recently been demonstrated to decrease the mitogenic activity of intracellular bFGF. Loss of this bioactivity has been implicated in impaired wound healing and microangiopathies of diabetes mellitus. In addition to intracellular localization, bFGF is also widely distributed in the extracellular matrix, primarily bound to heparan sulphate proteoglycans (HSPGs). Nonetheless, it is not clear if non-enzymic glycosylation similarly inactivates matrix-bound bFGF. To investigate this, we measured the effect of non-enzymic glycosylation on bFGF bound to heparin, heparan sulphate and related compounds. Incubation of bFGF with the glycosylating agents glyceraldehyde 3-phosphate (G3P; 25 mM) or fructose (250 mM) resulted in loss of 90% and 40% of the mitogenic activity of bFGF respectively. Treatment with G3P and fructose also decreased the binding of bFGF to a heparin column. If heparin was added to bFGF prior to non-enzymic glycosylation, the mitogenic activity and heparin affinity of bFGF were nearly completely preserved. A similar protective effect was demonstrated by heparan sulphate, low-molecular-mass heparin and the polysaccharide dextran sulphate, but not by chondroitin sulphate. Whereas non-enzymic glycosylation of bFGF with G3P impaired its ability to stimulate c-myc mRNA expression in fibroblasts, no such impairment was noticeable when bFGF was glycosylated in the presence of heparin. Taken together, these results suggest that HSPG-bound bFGF is resistant to non-enzymic glycosylation-induced loss of activity. Therefore, alteration of this pool probably does not contribute to impaired wound healing seen in diabetes mellitus. (+info)
Competition of Abeta amyloid peptide and apolipoprotein E for receptor-mediated endocytosis.
The genetic polymorphism of apolipoprotein E (apoE) is associated with the age of onset and relative risk of Alzheimer's disease (AD). In contrast to apoE3, the wild type allele, apoE4 confers an increased risk of late-onset AD. We demonstrate that the beta-amyloid peptide isoforms Abeta (1-28), Abeta (1-40), and Abeta (1-43) compete for the cellular metabolism of apoE3 and apoE4 containing beta-very low density lipoproteins. An antibody raised against Abeta (1-28) cross-reacted with recombinant apoE. Epitope mapping revealed positive amino acid clusters as common epitopes of Abeta (13 through 17; HHQKL) and apoE (residues 144 through 148; LRKRL), both regions known to be heparin binding domains. Abeta in which amino acids 13 through 17 (HHQKL) were replaced by glycine (GGQGL) failed to compete with the cellular uptake of apoE enriched betaVLDL. These observations indicate that Abeta and apoE are taken up into cells by a common pathway involving heparan sulfate proteoglycans. (+info)