heparitin-sulfate and sodium-sulfate

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.

Topics: Animals; Binding Sites; Cell Adhesion; Cell Adhesion Molecules; Cell Line; CHO Cells; Chondroitin ABC Lyase; Chondroitin Sulfates; Cricetinae; Embryo, Mammalian; Fibrosarcoma; Gene Expression; Heparitin Sulfate; Humans; Immunoblotting; Immunosorbent Techniques; Kalinin; Keratinocytes; Kidney; Laminin; Membrane Glycoproteins; Polysaccharide-Lyases; Proteoglycans; Recombinant Proteins; Sulfates; Sulfur Radioisotopes; Syndecan-1; Syndecans; Transfection; Tumor Cells, Cultured

Topics: Animals; Chondroitin ABC Lyase; Chromatography; Glycosaminoglycans; Heparitin Sulfate; L Cells; Mice; Phosphoadenosine Phosphosulfate; Proteoglycans; Sulfates; Sulfur Radioisotopes

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.

Topics: Cell Adhesion; Electrophoresis, Gel, Two-Dimensional; Glycosaminoglycans; Heparitin Sulfate; Humans; Protein Conformation; Protein Processing, Post-Translational; Receptors, Fibronectin; Sulfates; Tumor Cells, Cultured

Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perl

Topics: Adolescent; Adult; Aged; Base Sequence; Cells, Cultured; Child; Child, Preschool; Chondroitin Sulfate Proteoglycans; Chromatography, High Pressure Liquid; DNA, Complementary; Enzyme-Linked Immunosorbent Assay; Epithelium; Glomerular Mesangium; Glucosamine; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Infant; Kidney Glomerulus; Leucine; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; Proteoglycans; RNA; RNA, Messenger; Sulfates; Sulfur Radioisotopes; Transcription, Genetic; Tritium

The kinetic parameters (Km and kcat) of human liver alpha-L-iduronidase were determined with a variety of heparin-derived disaccharide and tetrasaccharide substrates. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrates heparin and heparan sulphate were maintained, were hydrolysed with catalytic efficiencies up to 255 times that observed for the simplest disaccharide substrate to be hydrolysed. The major aglycone structure that influenced both substrate binding and enzyme activity was the presence of a C-6 sulphate ester on the residue adjacent to the iduronic acid residue being hydrolysed. Sulphate ions and a number of substrate and product analogues were potent inhibitors of enzyme activity. Human liver alpha-L-iduronidase activity towards 4-methylumbelliferyl alpha-L-iduronide at pH 4.8 had two Km values of 37 microM and 1.92 mM with corresponding kcat. values of 299 and 650 mol of product formed/min per mol of enzyme respectively, which may explain the wide range of Km values previously reported for alpha-L-iduronidase activity toward its substrate. Skin fibroblast alpha-L-iduronidase activity towards the heparin-derived oligosaccharides was influenced by the same substrate aglycone structural features as was observed for the human liver enzyme. A comparison was made of the effect of substrate aglycone structure upon catalytic activities of the enzymes which act to degrade the highly sulphated regions of heparan sulphate. A model was proposed whereby the substrate is directed from alpha-L-iduronidase to subsequent enzyme activities to ensure the efficient degradation of heparan sulphate.

Topics: Carbohydrate Sequence; Fibroblasts; Heparitin Sulfate; Humans; Hydrogen-Ion Concentration; Iduronidase; Kinetics; Lysosomes; Molecular Sequence Data; Skin; Sulfates

Cell surface proteoglycans of aortic smooth muscle cells of atherosclerosis-susceptible White Carneau (WC) and atherosclerosis-resistant Show Racer (SR) pigeons were compared to determine differences that may be involved in the greater proliferative properties of cultured WC cells. Using [35S]-sodium sulfate and [3H]-glucosamine as labeling precursors, chondroitin sulfate-proteoglycan (CS-PG) and heparin sulfate-proteoglycan (HS-PG) were identified as distinct molecules associated with the plasma membrane. Heparan sulfate-proteoglycan was reduced up to 50% in WC compared with SR cells, and, based on interaction with ion-exchange resin, had a lower charge density. These differences were not observed for the CS-PG from the two cell types. The mode of association of the cell surface PG with the plasma membrane was examined. Dissociation with 1 mol/l (molar) sodium chloride indicated that less than 10% of total cell surface PG were ironically associated with the cells. The remainder required detergent extraction, suggesting hydrophobic interactions with the plasma membrane. Both CS-PG and HS-PG displayed affinity for octyl sepharose and both were identified in isolated plasma membranes. These data present the first description of a hydrophobic CS-PG that is a significant and distinct cell-associated PG in arterial smooth muscle cells. The observation of decreased and structurally altered HS-PG in WC compared with SR cells is consistent with a potential growth regulatory function for this molecule.

Topics: Animals; Aorta; Cell Membrane; Cells, Cultured; Chondroitin Sulfates; Chromatography, Ion Exchange; Columbidae; Glucosamine; Heparitin Sulfate; Muscle, Smooth, Vascular; Sodium Radioisotopes; Sulfates; Tritium

Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J.G., and R.W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (approximately 70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, greater than 90% of the 35S-labeled material present was in this heparan sulphate proteoglycan.

Topics: Animals; Ascorbic Acid; Basement Membrane; Cell Fractionation; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Epithelium; Female; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Lens, Crystalline; Male; Proline; Proteoglycans; Rabbits; Sulfates; Sulfur Radioisotopes; Tritium

Rabbit lens epithelial cells synthesize and secrete a variety of [35S]sulphate-labeled glycoconjugates in vitro. Associated with the cell layer, and with the medium, was a high molecular weight glycoconjugate(s) that contained heparan sulphate which was apparently covalently linked to sulphated glycoprotein. This component(s) was eluted in the void volume of a Sepharose CL-2B column and could not be fractionated by detergent treatment or extraction with lipid solvents. The cell layer also contained glycosaminoglycans (72% heparan sulphate, 28% chondroitin sulphate), as well as a small proportion of a low molecular weight sulphated glycoprotein. The major 35S-labeled species secreted into the medium were sulphated glycoproteins with approximate molecular weights of 120,000 and 35,000 together with a heparan sulphate proteoglycan. This proteoglycan could be precipitated from the culture medium with 30% saturated (NH4)2SO4 and eluted from Sepharose CL-4B columns at approximately the same position (Kav = 0.15) as heparan sulphate proteoglycans described in the basement membrane of the EHS "sarcoma" (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin, 1980, Proc. Natl. Acad. Sci. USA, 77:4494-4498) and of the mouse mammary epithelium (David, G., and M. Bernfield, 1981, J. Cell Biol., 91:281-286). Its presence in the culture medium was unanticipated but may be explained by the inability of these cultures to deposit a basement membrane when grown on a plastic surface. The relationship of this heparan sulphate proteoglycan to the lens epithelial basement membrane is the subject of the following paper.

Topics: Animals; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Epithelium; Female; Glycoproteins; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Lens, Crystalline; Male; Molecular Weight; Rabbits; Sulfates; Sulfur Radioisotopes

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Chondroitin Sulfates; Chromatography, DEAE-Cellulose; Culture Media; Glucosamine; Glycosaminoglycans; Hematopoiesis; Heparitin Sulfate; Hyaluronic Acid; Mice; Molecular Weight; Sulfates

A gradual decline in the synthesis of glycosaminoglycans, as evidenced by reduced rates of incorporation of [35S] sulfate and [14C] glucosamine into cellular and medium glycosaminoglycans, was observed during the last (about 5) population doublings before phase-out. The decline was accompanied by a change in the distribution pattern of individual glycosaminoglycans with a relative decrease in the incorporation rate of [14C] glucosamine into cellular and medium hyaluronic acid. The incorporation rate of [14D] glucosamine and [35S] sulfate into cellular and medium heparan sulfate continually increased during the last population doublings ("senescence"). The possibility of a coupling between cell growth and hyaluronic acid synthesis or an involvement of hyaluronic acid in the adhesion of cells (among one another or/and to the substratum), and the functional significance of heparan sulfate as a growth inhibitor were discussed.

Topics: Aging; Cell Survival; Chondroitin Sulfates; Dermatan Sulfate; Fibroblasts; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Sodium; Sulfates