Proteoglycan Binding Sites Research Articles

Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion to fibronectin splice variants.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Metabolism of VLDL is increased in streptozotocin-induced diabetic rat hearts.

In streptozotocin (STZ)-induced diabetic rats, we previously showed an increased heparin-releasable (luminal) lipoprotein lipase (LPL) activity from perfused hearts. To study the effect of this enlarged LPL pool on triglyceride (TG)-rich lipoproteins, we examined the metabolism of very-low-density lipoprotein (VLDL) perfused through control and diabetic hearts. Diabetic rats had elevated TG levels compared with control. However, fasting for 16 h abolished this difference. When the plasma lipoprotein fraction of density <1.006 g/ml from fasted control and diabetic rats was incubated in vitro with purified bovine or rat LPL, VLDL from diabetic animals was hydrolyzed as proficiently as VLDL from control animals. Post-heparin plasma lipolytic activity was comparable in control and diabetic animals. However, perfusion of control and diabetic rats with heparinase indicated that diabetic hearts had larger amounts of LPL bound to heparan sulfate proteoglycan-binding sites. [(3)H]VLDL obtained from control rats, when recirculated through the isolated heart, disappeared at a significantly faster rate from diabetic than from control rat hearts. This increased VLDL-TG hydrolysis was essentially abolished by prior perfusion of the diabetic heart with heparin, implicating LPL in this process. These findings suggest that the enlarged LPL pool in the diabetic heart is present at a functionally relevant location (at the capillary lumen) and is capable of hydrolyzing VLDL. This could increase the delivery of free fatty acid to the heart, and the resultant metabolic changes could induce the subsequent cardiomyopathy that is observed in the chronic diabetic rat.

Elastase-mediated Release of Heparan Sulfate Proteoglycans from Pulmonary Fibroblast Cultures: A MECHANISM FOR BASIC FIBROBLAST GROWTH FACTOR (bFGF) RELEASE AND ATTENUATION OF bFGF BINDING FOLLOWING ELASTASE-INDUCED INJURY

We have investigated elastase-mediated alterations in the expression of basic fibroblast growth factor (bFGF) receptors and proteoglycan co-receptors and characterized the subsequent effects on bFGF receptor binding profiles. For these studies, pulmonary fibroblast cultures were treated with porcine pancreatic elastase, and elastase-mediated changes in bFGF receptor expression and binding profiles were assessed. Quantitation of [(35)S]sulfate-labeled proteoglycan and total glycosaminoglycan release from fibroblast matrices indicated that elastase treatment released sulfated proteoglycan from the cell surface in a time- and dose-dependent fashion that correlated strongly with elastase-mediated bFGF release. Ligand binding studies indicated that elastase treatment decreased total binding of (125)I-bFGF to the cell surface and affected both fibroblast growth factor receptor and heparan sulfate proteoglycan (HSPG) binding sites. Western blot analyses indicated that elastase treatment did not release significant amounts of fibroblast growth factor receptor protein. These findings indicate that elastase-mediated HSPG release from fibroblast matrices reduces the effective affinity of bFGF for its receptor. Collectively, these studies suggest that HSPG co-receptors are important mediators of the pulmonary fibroblast response to elastase treatment and that bFGF, HSPG, and other elastase-released entities play an important role in the response of the lung to chronic injury.

The NH2-terminal Region of Apolipoprotein B Is Sufficient for Lipoprotein Association with Glycosaminoglycans

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100- and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48- and apoB100- containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH2-terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH2-terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH2-terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH2-terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH2-terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH2-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

Identification of the principal proteoglycan-binding site in LDL. A single-point mutation in apo-B100 severely affects proteoglycan interaction without affecting LDL receptor binding.

The subendothelial retention of LDLs through their interaction with proteoglycans has been proposed to be a key process in the pathogenesis of atherosclerosis. In vitro studies have identified eight clusters of basic amino acids in delipidated apo-B100, the protein moiety of LDL, that bind the negatively charged proteoglycans. To determine which of these sites is functional on the surface of LDL particles, we analyzed the proteoglycan-binding activity of recombinant human LDL isolated from transgenic mice. Substitution of neutral amino acids for the basic amino acids residues in site B (residues 3359-3369) abolished both the receptor-binding and the proteoglycan-binding activities of the recombinant LDL. Chemical modification of the remaining basic residues caused only a marginal further reduction in proteoglycan binding, indicating that site B is the primary proteoglycan-binding site of LDL. Although site B was essential for normal receptor-binding and proteoglycan-binding activities, these activities could be separated in recombinant LDL containing single-point mutation. Recombinant LDL with a K3363E mutation, in which a glutamic acid had been inserted into the basic cluster RKR in site B, had normal receptor binding but interacted defectively with proteoglycans; in contrast, another mutant LDL, R3500Q, displayed defective receptor binding but interacted normally with proteoglycans. LDL with normal receptor-binding activity but with severely impaired proteoglycan binding will be a unique resource for analyzing the importance of LDL- proteoglycan interaction in atherogenesis. If the subendothelial retention of LDL by proteoglycans is the initial event in early atherosclerosis, then LDL with defective proteoglycan binding may have little or no atherogenic potential.

Developmentally regulated expression and localization of fibroblast growth factor receptors in the human muscle.

Fibroblast growth factors (FGFs) are believed to play a key role in tissue differentiation and maturation. Thus, the expression of the four members of the high-affinity tyrosine kinase FGF receptor family (FGFRs) and of the low-affinity heparan sulphate proteoglycan binding sites, syndecan-1 and perlecan, was studied in the human skeletal muscle during development. Northern blot analysis demonstrated a developmentally regulated expression of the mRNAs for FGFR-1, FGFR-3, FGFR-4, whereas only traces of FGFR-2 mRNA were found. Each receptor type had a different developmental pattern, suggesting an independent regulation. Signal for FGFR-3 was retained only in the adult muscle. Among the low-affinity FGF binding sites, perlecan was absent, whereas RNA transcript for syndecan-1 peaked at week 13 of gestation, after which a significant decrease was observed. Immunohistochemistry for FGFRs revealed that their localization changed with muscle maturation. At early embryonic stages, FGFR-3 and FGFR-4 had a scattered distribution in the tissue, and FGFR-1 was found on myotube and myofiber plasma membranes. At later stages, FGFR-1 positivity decreased and was found in a few areas of the muscle, FGFR-3 was concentrated in the nuclei of some, but not all, muscle fibers, and FGFR-4 maintained an association with plasma membrane. In adult tissue, weak positivity for FGFR-3 and FGFR-4 was observed in the connective tissue only. When immunocytochemistry was performed on human fetal myoblasts in culture, confocal microscope analysis revealed a nonhomogeneous cell membrane distribution of FGFRs. Taken together, the data strongly suggest that developmentally regulated expression and cell distribution of FGFRs play a role during muscle maturation.

Erratum: Endothelial binding sites for lipoprotein lipase are not diminished in perfused hearts from diabetic rats

This article was published with the wrong French abstract. The correct English and French abstracts are printed below in full. The Publisher regrets this error. Abstract : The possibility that diabetes reduces functional, heparin-releasable lipoprotein lipase (HR-LPL) activity on the coronary vasculature of perfused hearts by altering endothelial binding sites for the enzyme was examined by measuring the binding and subsequent heparin-induced release of exogenous lipoprotein lipase purified from bovine milk (mLPL). Rat hearts were first perfused with heparin (5 U/mL) for 5 min to displace endogenous HR-LPL into the perfusate. The subsequent perfusion of control hearts with 0.05-2 µg/mL mLPL resulted in a progressive increase in bound exogenous exzyme that could be released by a second heparin perfusion. Induction of an acute, insulin-deficient model of diabetes (100 mg/kg streptozotocin 4-5 days prior to heart perfusions) reduced endogenous HR-LPL activity, but the binding and heparin-induced release of mLPL (0.5 µg/mL) were the same as measured in control hearts. Therefore, diabetes does not alter low-affinity, high-capacity proteoglycan binding sites for mLPL on the endothelium of perfused hearts.Key words: diabetes, lipoprotein lipase, perfused hearts.

Peptide sequences in glutaraldehyde-linked proteodermatan sulphate: collagen fragments from rat tail tendon locate the proteoglycan binding sites.

Collagen fibrils associate noncovalently with proteodmman sulphates (PDSs) at specitic and regular sites in the gap zone, at the d or e band. We call these strudures 'shape modules' because they appear to organise the collagen fibril matrix [l]. A shape module is a quaternary structure, collagen fibril4DS protein core+ AGAG chaiwAGAG chairwPDS protein coreccollagen fibril. Inspection of collagen fibril aminoacid sequences in PDS binding regions implicated 1 l-residue stretches in a1 chains [2] vvhich marry wll to the structure of PDS in complementary binding sites [3]. Alternative wggastions [4] rely on much less specific data, especially in vitm models [5] that are capable of several interpetations [6]. The most relevant and valid approach is to investigate the tissue directly. Our aim WBS to prepare a fragment of collegen from tissue with PDS covalently attached that could be sequenced to determine the composition and position of this binding site within the collagen molecule. We used glutaraldehyde as the crosslinker since this fixes the proteoglycans at their specific binding sites [7]. Freeze dried rat tail tendon (RlT) was fixed in 0.2% glutaraldehyde solution at pH 6.5, reduced with 0.1M sodium borohydride, extracted with 2M MgC12 at 1OOOC and then digested with type VII clostridial collagenase at pH 7.4. The supernatant was precipitated with cetylpyridiniurn chloride (CPC) to recover PDS-rich species. Hydroxyproline and polyanion analyses, electrophoreses with Alcian blue [8] or Coomassie blue staining wre performed on deposits and extracts, before and after papain digestion. Aminoacid analyses and Edman sequencing w r e carried out on the PDS rich fractions. Controls were based on unfixed RlT. Fixed RTT remained >95% intact after hot MgC12 extraction compared with 40% of unfixed RTT. Over 90 % of the hydroxyproline from extracted fixed RTT was solubilised by collagenase digestion, the CPC product containing both polyanion and hydroxyproline. Edman sequencing of the CPC product revealed Gly as the only significant N terminal residue and further showed a sequence GAKGDRGET in a complex mixture which is characteristic of the a1 chain in the e band in the quarter stassered array. This finding is compatible with the model (31 in which PDS is associated at around residues 855-865 in the a1 chain. It is also compatible with one of four possible binding sites that are otherwise not convincingly differentiable, derived from in vitm models [5].

Abeta(1-40) prevents heparanase-catalyzed degradation of heparan sulfate glycosaminoglycans and proteoglycans in vitro. A role for heparan sulfate proteoglycan turnover in Alzheimer's disease.

Alzheimer's disease is characterized by senile plaques composed of polymeric fibrils of beta amyloid (Abeta), a 39-42-amino acid peptide formed after proteolytic processing of the amyloid precursor protein (betaAPP). Heparan sulfate proteoglycans have been shown to colocalize with Abeta in Alzheimer's disease brain, and experimental evidence indicates that the interactions between the proteoglycan and the peptide are important for the promotion, deposition, and/or persistence of the senile plaques. Studies in rat brain indicated that both the core protein and the heparan sulfate glycosaminoglycan chains are required for amyloid fiber formation and deposition in vivo (Snow, A. D., Sekiguchi, R., Nochlin, D., Fraser, P., Kimata, K. , Mizutani, A., Arai, M., Schreier, W. A., and Morgan, D. G. (1994) Neuron 12, 219-234), suggesting that one mechanism to prevent the formation of Abeta-heparan sulfate proteoglycan complexes that lead to deposition of amyloid would be to degrade the proteoglycan. Normally, heparan sulfate proteoglycans are internalized and degraded to short glycosaminoglycans by intracellular heparanases. These reactions occur in the endosomal-lysosomal pathway, which is the same intracellular location where betaAPP is processed to Abeta. Using partially purified heparanase activities from Chinese hamster ovary cells we examined whether Abeta(1-40) affects the catabolism of Chinese hamster ovary heparan sulfate glycosaminoglycans and proteoglycans in vitro. Abeta(1-40) binds to both the long heparan sulfate glycosaminoglycans attached to core proteins and the short, heparanase-derived chains in a concentration-dependent and pH-dependent manner. When Abeta(1-40) is added to heparanase assays, it prevents the partially purified activities from releasing heparan sulfate chains from core proteins and degrading them to short glycosaminoglycans; however, a large molar excess of the peptide to heparan sulfate is required to see the effect. Our results suggest that normally the levels of Abeta in the endosomal pathway are not sufficient to interfere with heparanase activity in vivo. However, once the level of Abeta-peptides are elevated, as they are in Alzheimer's disease, they could interact with heparan sulfate proteoglycans and prevent their catabolism. This could promote the formation and deposition of amyloid, since the binding of Abeta to the proteoglycan species will predominate.

The Fibrinogen-like Globe of Tenascin-C Mediates Its Interactions with Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β

Two nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan (the extracellular domain of protein-tyrosine phosphatase-zeta/beta), are high-affinity ligands of tenascin-C. Using portions of tenascin-C expressed as recombinant proteins in human fibrosarcoma cells, we have demonstrated both by direct radioligand binding assays and inhibition studies that phosphacan binding is retained in all deletion variants except those lacking the fibrinogen-like globe and that phosphacan binds to this single domain with nearly the same affinity (Kd approximately 12 nM) as to native or recombinant tenascin-C. However, maximum binding of neurocan requires both the fibrinogen globe and some of the adjacent fibronectin type III repeats. Binding of phosphacan and neurocan to intact tenascin-C, and of phosphacan to the fibrinogen globe, is significantly increased in the presence of calcium. Chondroitinase treatment of the proteoglycans did not affect their binding to either native tenascin-C or to any of the recombinant proteins, demonstrating that these interactions are mediated by the proteoglycan core proteins rather than through the glycosaminoglycan chains. These results are also consistent with rotary shadowing electron micrographs that show phosphacan as a rod terminated at one end by a globular domain that is frequently seen apposed to the fibrinogen globe in mixtures of phosphacan and tenascin-C. C6 glioma cells adhere to and spread on deletion variants of tenascin-C containing only the epidermal growth factor-like domains or the fibronectin type III repeats and the fibrinogen globe. In both cases cell adhesion was inhibited by similar concentrations of phosphacan, demonstrating that the fibrinogen globe is not necessary for this effect, which is apparently mediated by a direct action of phosphacan on the cells rather than by its interaction with the proteoglycan binding site on tenascin-C.

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

Endothelial binding sites for lipoprotein lipase are not diminished in perfused hearts from diabetic rats

The possibility that diabetes reduces functional, heparin-releasable lipoprotein lipase (HR-LPL) activity on the coronary vasculature of perfused hearts by altering endothelial binding sites for the enzyme was examined by measuring the binding and subsequent heparin-induced release of exogenous lipoprotein lipase purified from bovine milk (mLPL). Rat hearts were first perfused with heparin (5 U/mL) for 5 min to displace endogenous HR-LPL into the perfusate. The subsequent perfusion of control hearts with 0.05-2 micrograms/mL mLPL resulted in a progressive increase in bound exogenous enzyme that could be released by a second heparin perfusion. Induction of an acute, insulin-deficient model of diabetes (100 mg/kg streptozotocin 4-5 days prior to heart perfusions) reduced endogenous HR-LPL activity, but the binding and heparin-induced release of mLPL (0.5 microgram/mL) were the same as measured in control hearts. Therefore, diabetes does not alter low-affinity, high-capacity proteoglycan binding sites for mLPL on the endothelium of perfused hearts.

Cell-surface Expression of an Amino-terminal Fragment of Apolipoprotein B Increases Lipoprotein Lipase Binding to Cells

Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.

Mapping the heparin-binding sites on type I collagen monomers and fibrils.

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed assembly or restructuring of the collagenous extracellular matrix.

Homologous sequences in fibrillar collagens may be proteoglycan binding sites.

Homologous sequences in fibrillar collagens may be proteoglycan binding sites.

A glycoprotein expressed by human fibrous astrocytes is a hyaluronate-binding protein and a member of the CD44 family.

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an M(r) of 80 kDa on SDS PAGE. The M(r) of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.

Release of lipoprotein lipase from cardiac myocytes by low‐molecular weight heparin

Incubation of cardiac myocytes from rat heart with low-molecular weight heparin (LMWH; Mr approx. 3 kDa) for 30 min resulted in a concentration-dependent release of lipoprotein lipase (LPL) activity into the incubation medium. The release of lipoprotein lipase from cardiac myocytes isolated from both control and diabetic rat hearts induced by LMWH (10 micrograms/mL) following incubation times of 10 or 30 min was significantly greater than that produced by unfractionated heparin (10 and 30 micrograms/mL) or decavanadate (1 mM). Since LMWH released more LPL activity into the incubation medium than unfractionated heparin following a short (10 min) incubation time. LMWH is probably more effective in displacing LPL bound to heparan sulfate proteoglycan binding sites on the cell surface of cardiac myocytes.

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.

A Role for Pericellular Proteoglycan in the Binding of Thrombin or Antithrombin III by the Blood Vessel Endothelium? – The Effects of Proteoglycan-Degrading Enzymes and Glycosaminoglycan-Binding Proteins on 125I-Thrombin Binding by the Rabbit Thoracic Aorta In Vitro

Rabbit thoracic aorta segments were treated with either proteoglycan-degrading enzymes or with glycosaminoglycan-binding proteins to examine the nature of the endothelial and subendothelial binding sites of 125I-thrombin. Treatment (5-30 min) with enzymes (heparitinase, chondroitinases AC or ABC) caused a decrease in 125I-thrombin binding by the endothelium (30-70%) and by the subendothelial (intima-media) layer (20-50%); a low-specificity protease destroyed endothelial binding almost entirely and reduced binding to the subendothelium by approximately 60% over a similar period. Of the glycosaminoglycan-binding proteins, pretreatment of the aorta wall with protamine caused a 30% decrease in thrombin binding to the endothelium whereas lipoprotein lipase (present during 125I-thrombin uptake) decreased binding by up to 40%. Pretreatment with antithrombin III did not significantly affect binding of either 125I-thrombin or 125I-FPR-inactivated thrombin. In contrast to thrombin, 125I-antithrombin III was not readily uptaken by the aorta segments. These observations indicate that, whereas the minimal binding by 125I-antithrombin III probably does not involve endothelial proteoglycan, a strong case can be made for endothelial and subendothelial proteoglycan binding sites for thrombin.

Binding of hyaluronic acid oligosaccharides by cartilage proteoglycan

Summary Hyaluronic acid oligosaccharides containing five or more disaccharide units form 1:1 complexes with proteoglycan, binding strongly with an association constant K estimated to be at least 50 μM −1 . Smaller oligosaccharides appear also to bind, K being about 1 to 2 μM −1 . These results are consistent with the observed competitive displacement of macromolecular hyaluronic acid from proteoglycan binding sites by oligosaccharides containing no fewer than five disaccharide units.