Identification of extracellular matrix ligands for the heparan sulfate proteoglycan agrin.

Agrin is a major brain heparan sulfate proteoglycan which is expressed in nearly all basal laminae and in early axonal pathways of the developing central nervous system. To further understand agrin's function during nervous system development, we have examined agrin's ability to interact with several heparin-binding extracellular matrix proteins. Our data show that agrin binds FGF-2 and thrombospondin by a heparan sulfate-dependent mechanism, merosin and laminin by both heparan sulfate-dependent and -independent mechanisms, and tenascin solely via agrin's protein core. Furthermore, agrin's heparan sulfate side chains encode a specificity in interactions with heparin-binding molecules since fibronectin and the cell adhesion molecule L1 do not bind agrin. Surface plasmon resonance studies (BIAcore) reveal a high affinity for agrin's interaction with FGF-2 and merosin (2.5 and 1.8 nM, respectively). Demonstrating a biological significance for these interactions, FGF-2, laminin, and tenascin copurify with immunopurified agrin and immunohistochemistry reveals a partial codistribution of agrin and its ECM ligands in the chick developing visual system. These studies and our previous studies, showing that merosin and NCAM also colocalize with agrin, provide evidence that agrin plays a crucial role in the function of the extracellular matrix and suggest a role for agrin in axon pathway development.     

Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells

Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and is specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.     

Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.     

Chemoenzymatic synthesis of heparan sulfate and heparin

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays essential physiological and pathophysiological functions. The biosynthesis of HS involves a series of specialised sulfotransferases, an epimerase and glycosyl transferases. The availability of these enzymes offers a promising method to prepare HS polysaccharides and structurally defined oligosaccharides. Given the fact that chemical synthesis of large HS oligosaccharides is extremely difficult, preparation of HS using a chemoenzymatic approach has gained momentum. This review article summarises recent progress on the development of a chemoenzymatic approach to prepare HS and HS oligosaccharides.     

Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells.

Monensin is a monovalent metal ionophore that affects the intracellular translocation of secretory proteins at the level of trans-Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition is dose dependent and affects the ratio [35S]-sulfate/[3H]-hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular weight. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin-treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6-position of the glucosamine moiety. All other biosynthetic steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C-6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans-Golgi compartment.     

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.     

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.     

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

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.     

Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.     

Involvement of heparan sulfate proteoglycan in sensing hypotonic stress in bovine aortic endothelial cells

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (-30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca(2+) transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca(2+) transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.     

The binding of human betacellulin to heparin, heparan sulfate and related polysaccharides

Recombinant human betacellulin binds strongly to heparin, requiring of the order of 0.8 M NaCl for its elution from a heparin affinity matrix. This is in complete contrast to the prototypic member of its cytokine superfamily, epidermal growth factor, which fails to bind to the column at physiological pH and strength. We used a well-established heparin binding ELISA to demonstrate that fucoidan and a highly sulfated variant of heparan sulfate compete strongly for heparin binding. Low sulfated heparan sulfates and also chondroitin sulfates are weaker competitors. Moreover, although competitive activity is reduced by selective desulfation, residual binding to extensively desulfated heparin remains. Even carboxyl reduction followed by extensive desulfation does not completely remove activity. We further demonstrate that both hyaluronic acid and the E. coli capsular polysaccharide K5, both of which are unsulfated polysaccharides with unbranched chains of alternating N-acetylglucosamine linked beta(1-4) to glucuronic acid, are also capable of a limited degree of competition with heparin. Heparin protects betacellulin from proteolysis by LysC, but K5 polysaccharide does not. Betacellulin possesses a prominent cluster of basic residues, which is likely to constitute a binding site for sulfated polysaccharides, but the binding of nonsulfated polysaccharides may take place at a different site.     

Use of sulfated linked cyclitols as heparan sulfate mimetics to probe the heparin/heparan sulfate binding specificity of proteins

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.     

Modulation of vascular smooth muscle cells proteoglycan synthesis by the extracellular matrix

In this study, we investigated the effect of the extracellular matrix (ECM) secreted by vascular cells on proteoglycan (PG) synthesis by vascular smooth muscle cells in culture. PG synthesis of human aortic smooth muscle cells plated on plastic or the matrices derived from vascular endothelial cells, vascular smooth muscle cells, or THP-1 macrophages was characterized. Smooth muscle cell and macrophage matrices increased both secreted and cellular smooth muscle cells PG production by 2.5-fold to 3.9-fold, respectively, over plastic and endothelial cell matrix. Macrophage matrix was more potent than smooth muscle cell matrix in this regard. Selective enzymatic removal of chondroitin sulfates, collagen, and elastin from smooth muscle cell matrix enhanced the stimulation of PG synthesis, as did the removal of chondroitin sulfates from macrophage matrix. PG turnover rates were similar for smooth muscle cells plated on the three matrices. The newly synthesized PG from cultures plated on smooth muscle cell-, and macrophage-derived matrices had greater charge density, larger molecular size, and longer glycosaminoglycan chains than those from endothelial cell matrix cultures. These data show that the ECM plays a major role in modulating vascular smooth muscle cell PG metabolism in vitro.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Extracellular matrix heparan sulfate modulates endothelial cell susceptibility to Staphylococcus aureus

The ability of extracellular matrix heparan sulfate to alter the susceptibility of human endothelial cells to S. aureus was investigated. Endothelial cells grown on extracellular matrix synthesized by S. aureus-infected endothelial cells were more susceptible to subsequent staphylococcal infection than endothelial cells grown on the extracellular matrix synthesized by untreated endothelial cells. Endothelial cells were more susceptible to S. aureus infection when (1) grown on heparitinase-treated extracellular matrix that removed heparan sulfate chains, (2) grown on extracellular matrix produced by chlorate-treated endothelial cells that reduced sulfation in the matrix heparan sulfate proteoglycans, (3) grown on heparan sulfate purified from extracellular matrix elaborated by infected endothelial cells, and (4) endothelial cells were chlorate-treated and therefore expressed desulfated cellular heparan sulfate proteoglycans. Extracellular matrix produced by S. aureus-infected endothelial cells contained heparan sulfate proteoglycans with reduced sulfation. The altered extracellular matrix with reduced sulfated heparan sulfate proteoglycans signalled the uninfected endothelial cells to produce under sulfated cellular heparan sulfate proteoglycans that increased S. aureus adherence to the endothelial cells. J. Cell. Physiol. 173:102–109, 1997. © 1997 Wiley-Liss, Inc.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Association of cell surface heparan sulfate proteoglycans of Schwann cells with extracellular matrix proteins

The terminal differentiation of Schwann cells is dependent on contact with basement membrane. The present study was undertaken to investigate the role of cell surface heparan sulfate proteoglycans (HSPGs) in mediating Schwann cell responses to extracellular matrix contact. Phosphatidylinositol-specific phospholipase C-releasable cell surface HSPGs purified from cultures of neonatal rat Schwann cells were subjected to affinity chromatography on immobilized laminin and fibronectin. Binding of the HSPG to both affinity matrices was observed. The strength of the association, however, was sensitive to the ionic strength of the buffer. In 0.1 M Tris-HCl, HSPG binding was essentially irreversible whereas in physiological ionic strength buffer (e.g. 0.142 M NaCl, 10 mM Tris), weaker binding was detected as a delay in elution of the HSPG from the affinity columns. Further studies of HSPG-laminin binding suggested that the binding was mediated by the glycosaminoglycan chains of the proteoglycans. Results of equilibrium gel filtration chromatography provided additional evidence for a reversible association of the HSPG and laminin with a Kd of approximately 1 x 10(-6) M. When Schwann cells were plated on plastic dishes coated with laminin, the cells attached and extended long slender processes. Inclusion of heparin, but not chondroitin sulfate, in the assay medium resulted in partial inhibition of process extension, but at concentrations of heparin which were higher than that needed to disrupt laminin-HSPG association in vitro. Addition of anti-integrin receptor antibodies resulted in more extensive inhibition of laminin-dependent process extension. Anti-integrin antibodies plus heparin essentially totally inhibited laminin-dependent process extension. These results demonstrate that cell surface HSPGs are capable of reversible association with extracellular matrix molecules and suggest that HSPG-laminin interactions play a role in laminin-dependent Schwann cell spreading.     

High concentrations of low density lipoprotein decrease basement membrane-associated heparan sulfate proteoglycan in cultured endothelial cells

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.     

Identification of a lipid-anchored heparan sulfate proteoglycan in Schwann cells

Schwann cells synthesize both hydrophobic and peripheral cell surface heparan sulfate proteoglycans (HSPGs). Previous analysis of the kinetics of radiolabeling suggested the peripheral HSPGs are derived from the membrane-anchored forms (Carey, D., and D. Evans. 1989. J. Cell Biol. 108:1891-1897). Peripheral cell surface HSPGs were purified from phytic acid extracts of cultured neonatal rat sciatic nerve Schwann cells by anion exchange, gel filtration, and laminin-affinity chromatography. Approximately 250 micrograms of HSPG protein was obtained from 2 X 10(9) cells with an estimated recovery of 23% and an overall purification of approximately 2000-fold. SDS-PAGE analysis indicated the absence of non-HSPG proteins in the purified material. Analysis of heparinase digestion products revealed the presence of at least six core protein species ranging in molecular weight from 57,000 to 185,000. The purified HSPGs were used to produce polyclonal antisera in rabbits. The antisera immunoprecipitated a subpopulation of 35SO4- labeled HSPGs that were released from Schwann cells by incubation in medium containing phosphatidylinositol-specific phospholipase C (PI- PLC); smaller amounts of immunoprecipated HSPGs were also present in phytic acid extracts. In the presence of excess unlabeled PI-PLC- released proteins, immunoprecipitation of phytic acid-solubilized HSPGs was inhibited. SDS-PAGE analysis of proteins immunoprecipitated from extracts of [35S]methionine labeled Schwann cells demonstrated that the antisera precipitated an HSPG species that was present in the pool of proteins released by PI-PLC, with smaller amounts present in phytic acid extracts. Nitrous acid degradation of the immunoprecipitated proteins produced a single 67,000-Mr core protein. When used for indirect immunofluorescence labeling, the antisera stained the external surface of cultured Schwann cells. Preincubation of the cultures in medium containing PI-PLC but not phytic acid significantly reduced the cell surface staining. The antisera stained the outer ring of Schwann cell membrane in sections of adult rat sciatic nerve but did not stain myelin or axonal membranes. This localization suggests the HSPG may play a role in binding the Schwann cell plasma membrane to the adjacent basement membrane surrounding the individual axon-Schwann cell units.