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.     

Immunohistochemical localization of heparan sulfate proteoglycan in human gastrointestinal tract

Heparan sulfate proteoglycans (HS-PGs) are associated with important cell functions, for example, cell motility, cell adhesion, and oncogenesis. We examined the localization of HS-PGs in normal and carcinoma tissues of the gastrointestinal tract to help elucidate their roles in these organs. Fresh surgical materials from 134 patients with carcinoma of the stomach or large intestine and 26 patients with various diseases of the small intestine were immunostained after fixation with 10E4 (an antibody against the HS of HS-PG) as a primary antibody. Immunoelectron microscopy (immunogold method) was also performed. The basolateral surfaces of normal tissues of the large and small intestines were strongly stained with antibody confirmed by electron microscopy. In the stomach, lesions with intestinal metaplasia showed the same staining as the intestines, although normal gastric tissue showed staining only in some parts of the basal layer of fundic and pyloric glands. Carcinoma tissues in all cases examined showed staining with antibody. Better results were obtained after fixation in acetic alcohol or zinc-containing solutions than in ordinary formalin. These characteristic localizations of HS-PG in intestines and stomachs suggest that this kind of HS-PG staining could be a hallmark characteristic of the intestine.     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Defects in eye development in transgenic mice overexpressing the heparan sulfate proteoglycan agrin

The importance of heparan sulfate proteoglycans (HSPGs) in neurodevelopment is becoming increasingly clear. However, studies on HSPGs are hampered by pleiotropic effects when synthesis or modification of heparan sulfate itself is targeted, and by redundancy when the core proteins are altered. Gain-of-function experiments can sometimes circumvent these issues. Here we establish that transgenic mice overexpressing the HSPG agrin have severe ocular dysgenesis. The defects occur through a gain-of-function mechanism and penetrance is dependent on agrin dosage. The agrin-induced developmental defects are highly variable, and include anophthalmia, persistence of vitreous vessels, and fusion of anterior chamber structures. A frequently observed defect is an optic stalk coloboma leading to the misdifferentiation of the optic stalk as retina, which becomes continuous with the forebrain. The defects in optic-stalk differentiation correlate with reduced sonic hedgehog immunoreactivity and overexpansion of the PAX6 domain from the retina into the optic stalk. The ocular phenotypes associated with agrin overexpression are dependent on genetic background, occurring with high penetrance in inbred C57BL/6J mice. Distinct loci sensitizing C57BL/6J mice to agrin-induced dysgenesis were identified. These results indicate that agrin overexpression will provide a tool to explore the molecular interactions of the extracellular matrix and cell surface in eye development, and provide a means for identifying modifier loci that sensitize mice to developmental eye defects.     

Fatty acylation of heparan sulfate proteoglycan from human colon carcinoma cells

A number of transmembrane proteins have been recently reported to be modified by the covalent addition of saturated fatty acids which may contribute to membrane targeting and specific protein-lipid interactions. Such modifications have not been reported in cell-associated heparan sulfate proteoglycans, although these macromolecules are known to be hydrophobic. Here, we report that a cell surface heparan sulfate proteoglycan is acylated with both myristate and palmitate, two long-chain saturated fatty acids. When colon carcinoma cells were labeled with [3H]myristic acid, a significant proportion of the label was shown to be specifically incorporated into the protein core of the proteoglycan. Characterization of fatty acyl moiety in the purified proteoglycan by reverse-phase high pressure liquid chromatography revealed that approximately 60% of the covalently bound fatty acids was myristate. We further show that this relatively rare 14-carbon fatty acid was bound to the protein core via a hydroxylamine- and alkali-resistant amide bond. The remaining 40% was the more common 16-carbon palmitate, which was bound via a hydroxylamine- and alkali-sensitive thioester bond. Palmitate appeared to be added post-translationally and derived in part from intracellular elongation of myristate, a process that occurred within the first two hours and was insensitive to inhibition of protein synthesis. Acylation of heparan sulfate proteoglycan represents a novel modification of this gene product and could play a role in a number of biological functions including specific interactions with membrane receptors and ligand stabilization.     

Presence of heparan sulfate proteoglycan in thyroid tissue

Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer containing 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparin sulfate proteoglycan in thyroid tissue for the first time.     

Leukocyte motility in response to neuropeptides is heparan sulfate proteoglycan dependent

Activation of neuropeptide receptors on leukocytes induces chemotaxis. We determined in Boyden chambers with micropore filters, whether in human monocytes and lymphocytes this migratory response is heparan sulfate proteoglycan (HSPG) dependent. Chemotaxis toward calcitonin gene-related peptide, secretoneurin, vasoactive intestinal peptide (VIP), and substance P (SP) was abolished by removal of heparan sulfate side chains from cell surface proteoglycans or by addition of anti-syndecan-4 antibodies. Inhibition of neuropeptide-induced chemotaxis by dimethyl sphingosine (DMS), an inhibitor of sphingosine kinase, indicates transactivation of the sphingosine-1-phosphate chemotaxis pathway which was previously identified as being syndecan-4-related. Data suggest that HSPGs are involved in neuropeptide-induced chemotaxis of leukocytes.     

Hemostatic properties and serum lipoprotein binding of a heparan sulfate proteoglycan from bovine aorta

The biologic properties of two major proteoglycans of bovine aorta, heparan sulfate proteoglycan and chondroitin sulfate-dermatan sulfate proteoglycan were compared. The heparan sulfate proteoglycan was isolated either by elastase digestion or by 4.0 M guanidine hydrochloride extraction, of aorta tissue, fractionated by CsCl isopycnic centrifugation and purified by chondroitinase ABC treatment. The first method resulted in considerably greater yield (about 70% of the total heparan sulfate proteoglycan of the tissue) than the second procedure (12% of total). The chondroitin sulfate-dermatan sulfate proteoglycan was obtained by 4.0 M guanidine-HCl extraction of aorta tissue followed by CsCl isopycnic centrifugation. The chemical composition of both heparan sulfate proteoglycan preparations was similar. Unlike the chondroitin sulfate-dermatan sulfate proteoglycan, which eluted in the void volume of Sepharose CL-6B column, the heparan sulfate proteoglycan preparations were each resolved into a high molecular weight fraction (kav = 0.18 and 0.13) and a low molecular weight fraction (kav = 0.47 and 0.36). The heparan sulfate proteoglycan preparations exhibited significantly more potent anticoagulant and platelet aggregation inhibitory activities than the chondroitin sulfate-dermatan sulfate proteoglycan. The protein core of the proteoglycan molecules did not seem to be essential for their hemostatic properties. The complex forming ability of the heparan sulfate proteoglycan with serum low density lipoproteins (LDL) was much less than that of chondroitin sulfate-dermatan sulfate proteoglycan in the presence and absence of Ca2+. Interaction between heparan sulfate proteoglycan and LDL was also much more sensitive to changes in the ionic strength of the medium than that of chondroitin sulfate-dermatan sulfate proteoglycan and the lipoprotein. Since the total sulfate content of both proteoglycans is almost similar, the smaller molecular size and hence the lower overall charge density of the heparan sulfate proteoglycan appears to be partly responsible for its low affinity for LDL. The differences in biologic properties of the two proteoglycans might have implications in the pathophysiology of cardiovascular diseases.     

A conformational change in heparan sulfate 3-O-sulfotransferase-1 is induced by binding to heparan sulfate

The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.     

Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts

Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell- associated forms act as precursors for the nonhydrophobic medium- released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell- associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.     

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.     

Estradiol-stimulated turnover of heparan sulfate proteoglycan in mouse uterine epithelium

Heparan sulfate proteoglycans (HSPGs) and dermatan sulfate/chondroitin sulfate proteoglycans may be extracted from the uterine epithelium of immature mice by a 1-min exposure of the luminal surface of excised uteri to 1% Nonidet P-40 detergent. In mice that are treated with estradiol there is a marked increase in free heparan sulfate glycosaminoglycan in the extract. (a) By Sepharose exclusion chromatography the [35S]sulfate-labeled major HSPG had a nominal Mr of 200-250 X 10(3), consisting of a core protein of about 80-90 X 10(3) Mr with about 8-10 heparan sulfate glycosaminoglycan chains (Mr = 13 X 10(3)). The HSPG had a lower bouyant density (less than 1.45 g/ml) than the dermatan sulfate/chondroitin sulfate proteoglycan and was heterogeneous, as was evident in the fact that HSPG attained equilibrium over a wide range of CsCl densities and also showed nonuniform interaction with octyl-Sepharose. (b) Virtually all of the major HSPG was removed when the epithelium was isolated by proteolysis, indicating a cell surface localization. A smaller, less prominent HSPG (nominal Mr = 80 X 10(3)) was synthesized during the first 2 h after isolation. (c) Label and chase experiments with and without chloroquine showed that virtually all of the free heparan sulfate glycosaminoglycan chains derived from endocytosis and lysosomal degradation of the plasma membrane-associated HSPG. We conclude that estradiol stimulates endocytosis of HSPG, predominantly from the basolateral epithelial surface and suggest that this HSPG turnover may reflect changes associated with blastocyst attachment and invasion of the endometrium.     

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.     

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.     

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.     

Apolipoprotein E receptor binding versus heparan sulfate proteoglycan binding in its regulation of smooth muscle cell migration and proliferation

This study showed that synthetic peptides containing either a single copy or tandem repeat of the receptor binding domain sequence of apolipoprotein (apo) E, or a peptide containing its C-terminal heparin binding domain, apoE-(211-243), were all effective inhibitors of platelet-derived growth factor (PDGF)-stimulated smooth muscle cell proliferation. In contrast, only the peptide containing a tandem repeating unit of the receptor binding domain sequence of apoE, apoE-(141-155)(2), was capable of inhibiting PDGF-directed smooth muscle cell migration. Peptide containing only a single unit of this sequence, apoE-(141-155), or the apoE-(211-243) peptide were ineffective in inhibiting PDGF-directed smooth muscle cell migration. Additional experiments showed that reductively methylated apoE, which is incapable of receptor binding yet retains its heparin binding capability, was equally effective as apoE in inhibiting PDGF-stimulated smooth muscle cell proliferation. However, reductively methylated apoE was unable to inhibit smooth muscle cell migration toward PDGF. Additionally, the receptor binding domain-specific apoE antibody 1D7 also mitigated the anti-migratory properties of apoE on smooth muscle cells. Finally, pretreatment of cells with heparinase failed to abolish apoE inhibition of smooth muscle cell migration. Taken together, these data documented that apoE inhibition of PDGF-stimulated smooth muscle cell proliferation is mediated by its binding to heparan sulfate proteoglycans, while its inhibition of cell migration is mediated through apoE binding to cell surface receptors.     

Immunocytochemical localization of heparan sulfate proteoglycan in human decidual cell secretory bodies and placental fibrinoid

A distinct ultrastructural feature of human decidual cells is the presence of membrane-bound secretory bodies, 0.3-0.5 micron in diameter, located within club-shaped processes at the cell periphery. These secretory bodies contain 30-60 nm electron-dense granules. Using specific antibody and the protein A-gold technique, we examined the localization of heparan sulfate proteoglycan in human decidual cells. Morphometric analysis of gold particles in cellular compartments was performed with a Zeiss Videoplan computer system. Immuno-gold staining was present in the decidual cell cytoplasm and the extracellular space, especially in the zone of the external lamina. Gold particles, indicating the locale of heparan sulfate proteoglycan, were concentrated over the electron-dense granular material within decidual secretory bodies contained in club-shaped processes at the cell periphery. Immunolabeling of placental fibrinoid was also observed. This report provides the first identification of a specific molecular constituent of decidual secretory bodies and indicates a role for these structures in secretion of the peri-decidual cell extracellular matrix.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

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.