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Characterization and N-terminal sequence of a heparan sulphate proteoglycan synthesized by endothelial cells in culture.

ABSTRACT: We have isolated from the conditioned medium of an established endothelial cell line a heparan sulphate proteoglycan whose involvement in the inhibition of the extrinsic coagulation pathway was reported in previous studies [Colburn & Buonassisi (1982) Biochem. Biophys. Res. Commun. 104, 220-227]. The proteoglycan was purified by gel filtration and ion-exchange chromatography, and appears to be free of contaminating proteins as determined by polyacrylamide-gel electrophoresis of the radioiodinated protein core before and after removal of the glycosaminoglycan chains by treatment with heparitinase. By this procedure the Mr of the protein core was estimated to be 22000. The N-terminal end was sequenced up to amino acid 25. The 21st residue is likely to be glycosylated. Analysis of the purified proteoglycan by gel-filtration chromatography yielded Kd values of 0.2 for the whole molecule and 0.35 for the glycosaminoglycan chains. The structure that emerges from these data is that of a heparan sulphate proteoglycan characterized by a relatively small protein core and few glycosaminoglycan chains.

SUBMITTER: Castillo CJ

PROVIDER: S-EPMC1148467 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Cell surface chondroitin sulphate proteoglycan 4 (CSPG4) binds to the basement membrane heparan sulphate proteoglycan, perlecan, and is involved in cell adhesion.

Project description:Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented. In contrast, the CSPG4 expressed by a colon carcinoma cell line, WiDr, was predominantly as a protein core on the cell surface lacking glycosaminoglycan (GAG) chains. This study demonstrated that CSPG4 immunopurified from the melanoma cell lines formed a complex with perlecan synthesized by the same cultured cells. Mechanistic studies showed that CSPG4 bound to perlecan via hydrophobic protein-protein interactions involving multiple sites on perlecan including the C-terminal region. Furthermore, this study revealed that CSPG4 interacted with perlecan to support cell adhesion and actin polymerization. Together these data suggest a novel mechanism by which CSPG4 expressing cells might attach to perlecan-rich matrices so as those found in connective tissues and basement membranes.

| S-EPMC5905647 | biostudies-literature

Purification and characterization of heparan sulphate proteoglycan from bovine brain.

Project description:A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of >200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after beta-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected.

| S-EPMC1220693 | biostudies-other

Isolation and characterization of dermatan sulphate and heparan sulphate proteoglycans from fibroblast culture.

Project description:35SO42(-)- and [3H]leucine-labelled proteoglycans were isolated from the medium and cell layer of human skin fibroblast cultures. Measures were taken to avoid proteolytic modifications during isolation by adding guanidinium chloride and proteolysis inhibitors immediately after harvest. The proteoglycans were purified and fractionated by density-gradient centrifugation, followed by gel and ion-exchange chromatography. Our procedure permitted the isolation of two major proteoglycan fractions from the medium, one large, containing glucuronic acid-rich dermatan sulphate chains, and one small, containing iduronic acid-rich ones. The protein core of the latter proteoglycan had an apparent molecular weight of 47000 as determined by polyacrylamide-gel electrophoresis, whereas the protein core of the former was considerably larger. The major dermatan sulphate proteoglycan of the cell layer was similar to the large proteoglycan of the medium. Only small amounts of the iduronic acid-rich dermatan sulphate proteoglycan could be isolated from the cell layer. Instead most of the iduronic acid-rich glycans appeared as free chains. The heparan sulphate proteoglycans found in the cell culture were largely confined to the cell layer. This proteoglycan was of rather low buoyant density and seemed to contain a high proportion of protein. The major part of the heparan sulphate proteoglycan from the medium had a higher buoyant density and contained a smaller amount of protein.

| S-EPMC1163073 | biostudies-other

Characterization of Heparan Sulfate Proteoglycan-positive Recycling Endosomes Isolated from Glioma Cells.

Project description:BACKGROUND:Heparan sulfate proteoglycans (HSPGs)-dependent endocytic events have been involved in glioma progression. Thus, comprehensive understanding of the intracellular trafficking complexes formed in presence of HSPGs would be important for development of glioma treatments. MATERIALS AND METHODS:Subcellular fractionation was used to separate vesicles containing HSPGs from the rat C6 glioma cell line. Isolated HSPG-positive vesicles were further characterized with liquid chromatography-mass spectrometry. RESULTS:The HSPG-positive vesicular fractions, distinct from plasma membrane-derived material, were enriched in endocytic marker, Rab11. Proteomic analysis identified more than two hundred proteins to be associated with vesicular membrane, among them, over eighty were related to endosomal uptake, recycling or vesicular transport. CONCLUSION:Part of HSPGs in glioma cells is internalized through clathrin-dependent endocytosis and undergo recycling. The development of compounds regulating HSPG-mediated trafficking will likely enable design of effective glioma treatment.

| S-EPMC5219918 | biostudies-literature

Heparan sulfate proteoglycan mediates shear stress-induced endothelial gene expression in mouse embryonic stem cell-derived endothelial cells.

Project description:It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived ECs. However, the underlying mechanisms mediating shear stress effects in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived EC were subjected to shear stress (5 dyn/cm(2)) for 8 h with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular EC-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it did not alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.

| S-EPMC3228881 | biostudies-literature

Domain structure of endothelial heparan sulphate.

Project description:The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. 'Inner' chain fragments, remaining on the protein core, were separated from 'outer' fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of 'inner' and 'outer' chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded 'inner' and 'outer' fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the 'outer' fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.

| S-EPMC1151520 | biostudies-other

Identification of a new membrane-bound heparan sulphate proteoglycan.

Project description:The morphological changes that occur during intestinal development have been extensively described, but the molecular basis of these changes is largely unknown. As a result of our efforts to identify molecules that play a role in intestinal morphogenesis during development, we have previously isolated a cDNA that is developmentally regulated in the intestine. This cDNA, named OCI-5, was recently shown to have 20-25% identity at the protein-sequence level with glypican and cerebroglycan, two heparan sulphate proteoglycans (HSPG) that are attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Here we provide experimental evidence indicating that OCI-5 is also a GPI-linked HSPG. We demonstrate this by showing that OCI-5 can be labelled with radioactive sulphate and can be digested by heparitinase, but not by chondroitinase. We also show that treatment with phosphatidylinositol-specific phospholipase C releases OCI-5 from the cell surface of COS cells transfected with an OCI-5 expression vector. The identification of OCI-5 as a GPI-linked HSPG confirms that this proteoglycan belongs to the same family of HSPGs that include glypican and cerebroglycan.

| S-EPMC1136036 | biostudies-other

Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.

Project description:Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

| S-EPMC1149861 | biostudies-other

Characterization and N-terminal sequence of human platelet proteoglycan.

Project description:Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed.

| S-EPMC1135341 | biostudies-other

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions.

Project description:Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.

| S-EPMC4210653 | biostudies-literature

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