Defining the interleukin-8-binding domain of heparan sulfate

Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans. It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes. By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate. We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate. These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of 相似文献   

Age-dependent modulation of heparan sulfate structure and function

Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.     

Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants

We have demonstrated by affinity chromatography that hepatocyte growth factor (HGF) binds strongly to heparan sulfate (HS). This substantiates previous suggestions that cell-surface heparan sulfate proteoglycans constitute the so-called low affinity cellular binding sites for HGF. Using a recombinant human HGF affinity column, we have analyzed the effects of various specific chemical and enzymatic modifications/depolymerizations of HS on its affinity in order to elucidate the polysaccharide structural determinants. Interaction is shown to be only slightly affected by digestion with heparinase I or III or by replacement of N-sulfates with N-acetyl groups. This suggests a specific role for sulfated domains containing nonsulfated IdceA residues, with only a small contribution from N-sulfates and IdceA(2-OSO3) residues. In addition, disaccharide analyses of various HGF-binding oligosaccharides indicate that affinity is more closely associated with 6-O-sulfation of GlcNSO3 residues than with sulfation at any other position. Although interaction can be demonstrated with heparinase III-resistant oligosaccharides as small as hexasaccharides, the highest affinity was found with oligosaccharides containing a minimum of 10-12 monosaccharides. The structural specificity of the HGF-HS interaction is thus shown to be radically different from that previously described for the basic fibroblast growth factor-HS interaction.     

N-Acetylated domains in heparan sulfates revealed by a monoclonal antibody against the Escherichia coli K5 capsular polysaccharide. Distribution of the cognate epitope in normal human kidney and transplant kidney with chronic vascular rejection

The Escherichia coli K5 capsular polysaccharide has the same (GlcUA-->GlcNAc)n structure as the nonsulfated heparan sulfate/heparin precursor polysaccharide. A monoclonal antibody (mAb 865) against the K5 polysaccharide has been described (Peters, H., Jürs, M., Jann, B., Jann, K., Timmis, K. N., and Bitter-Sauermann, D. (1985) Infect. Immun. 50, 459-466). In this report, we demonstrate the binding of anti-K5 mAb 865 to N-acetylated sequences in heparan sulfates and heparan sulfate proteoglycans but not to heparin. This is shown by direct binding and fluid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assay. In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide. By testing chemically modified K5 and heparin polysaccharides, we found that each of the modifications that occur during heparan sulfate (HS) synthesis (N-sulfation, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865. Samples of heparan sulfate from human aorta (HS-II) were selectively degraded so as to allow the separate isolation of N-sulfated and N-acetylated block structures. N-Sulfated oligosaccharides (obtained after N-deacetylation by hydrazinolysis followed by nitrous acid deamination at pH 3.9) were not recognized by mAb 865, in contrast to N-acetylated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II. Analysis of the latter's pH 1.5 deamination products by gel filtration indicated that a minimal size of 18 saccharide units was necessary for antibody binding. These results lead us to propose bivalent antibody-heparan sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (>/=18 saccharide units) N-acetylated domain and additionally with single epitopes present in two N-acetylated sequences (each <18 saccharide units) bridged by a short N-sulfated domain. Immunohistochemistry with mAb 865 on cryostat sections of normal human kidney tissue, revealed its binding to most but not all renal basement membranes. However, all renal basement membranes contain heparan sulfate, as shown by a mAb against heparitinase-digested heparan sulfate stubs (mAb 3G10). This finding indicates that not all heparan sulfate chains present in basement membranes express the mAb 865 epitopes. Besides the normal distribution, mAb 865 staining was found in fibrotic and sclerotic lesions in vessels, interstitium, and mesangium in transplant kidneys with chronic vascular rejection. Occasionally, a decrease of staining was observed within tubulo-interstitium and glomeruli. These findings show that N-acetylated sequences in heparan sulfates can be demonstrated by anti-K5 mAb 865 in normal and diseased kidneys.     

Structural studies of octasaccharides derived from the low-sulfated repeating disaccharide region and octasaccharide serines derived from the protein linkage region of porcine intestinal heparin

Four octasaccharide serines and three octasaccharides were isolated after heparinase treatment of porcine intestinal heparin. Their structures were characterized by enzymatic digestion in conjunction with HPLC and 500 MHz 1H NMR spectroscopy. Three of the four octasaccharide serines were structurally identical with those isolated previously, whereas one has the unreported structure DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4GlcAbe ta1-4GlcNAca lpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta 1-O-Ser (DeltaHexA, GlcN, IdceA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, respectively). The other three octasaccharides were isolated for the first time as discrete structures and shared the common core hexasulfated sequence DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4IdceAa lpha1-4GlcNA calpha1-4GlcAbeta1-4GlcN(N-sulfate)alpha1-4IdceA (2-sulfate)alpha1-4Gl cN(N,6-disulfate) with one or two additional sulfate groups. The octasaccharides which were derived from the low-sulfated repeating disaccharide region of heparin contained the common trisaccharide sequence -4IdceAalpha1-4GlcNAcalpha1-4GlcAbeta1- [Yamada, S., Yamane, Y., Tsuda, H., Yoshida, K., and Sugahara, K. (1998) J. Biol. Chem. 273, 1863-1871], suggesting the programmed biosynthesis of heparin. These octasaccharides are the largest oligosaccharides isolated so far from the low-sulfated irregular region of heparin. Since oligosaccharides larger than a pentasaccharide appear to potentially exhibit binding activities toward growth factors or other functional proteins, they will be useful for investigating the structural requirement for molecular interactions between heparin and/or heparan sulfate and biologically active proteins. During the course of the present structural studies, we evaluated the NMR data accumulated thus far on heparin oligosaccharides and found several interesting rules on chemical shifts of proton signals affected by the neighboring sugar residues and their sulfation, which will be in turn useful for determining structures of unknown heparin and/or heparan sulfate oligosaccharides based on the proton resonances.     

Introduction: social cognition, psychodynamic psychology, and the representation and processing of emotionally significant information

The biological activity of basic fibroblast growth factor (bFGF) is influenced greatly by direct binding to heparin and heparan sulphate (HS). Heparin-derived oligosaccharides have been utilized to determine the structural requirements present in the polymer that account for binding to bFGF. We had previously demonstrated that fragments > 6 mer can inhibit the interaction between cell surface heparan sulphate proteoglycan (HSPG) and bFGF, and bFGF-induced proliferation of adrenocortical endothelial (ACE) cells. In contrast, oligosaccharides > 10 mer can enhance the binding of bFGF to its high-affinity receptor or support bFGF-induced mitogenesis in ACE cells (Ishihara et al., J. Biol. Chem., 268, 4675-4683, 1993). We have extended these studies to size- and structure-defined oligosaccharides from heparin, 2-O-desulphated (2-O-DS-) heparin, 6-O-desulphated (6-O-DS-) heparin, carboxy-reduced (CR-) heparin and carboxy-amidomethylsulphonated (AMS-) heparin. Oligosaccharides from these polymers were fractionated on a bFGF-affinity column and were assessed as inhibitors or enhancers of specific bFGF-derived biological activities. The results of these studies indicate that both 2-O-sulphate and the negative charge of the carboxy group [L-iduronic acid (IdoA) residues] are required for specific interactions of heparin-derived oligosaccharides with bFGF and for modulation of bFGF mitogenic activity. In addition, the charge of the carboxy groups in uronic acids can be replaced by other functional groups with a negative charge, such as the amidomethyl sulphonate moiety described here.     

Novel sulfated oligosaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K. Unexpected degradation by chondroitinase ABC

We prepared a series of oligosaccharides from king crab cartilage chondroitin sulfate K after exhaustive digestion with testicular hyaluronidase, and determined the structures of four tetrasaccharides and a pentasaccharide by fast atom bombardment mass spectrometry, high performance liquid chromatography analysis of chondroitinase AC-II digests, and 500-MHz 1H NMR spectroscopy. The tetrasaccharides shared the common core structure GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3GalNAc with various sulfation profiles. One structure was GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), whereas three of them have the following hitherto unreported structures including a novel glucuronate 3-O-sulfate: GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), GlcAbeta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), and GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), where 3S or 4S represents 3-O- or 4-O-sulfate, respectively. The structure of the pentasaccharide was determined as GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1- 3GalNAc(4S)beta1-4GlcA. Chondroitinase ABC digestion of the tetrasaccharides with GlcA(3S) at the internal position destroyed the disaccharide unit containing GlcA(3S) derived from the reducing side and resulted in only the disaccharide unit from the non-reducing side. In contrast, these tetrasaccharides remained totally resistant to chondroitinase AC-II. The results indicated that it is necessary to reevaluate the disaccharide composition of chondroitin sulfate poly- or oligosaccharides purified from various biological sources, since they were usually determined after chondroitinase ABC digestion. It is probable that the structures containing GlcA(3S) would not have been detected.     

Interaction between pseudorabies virus and heparin/heparan sulfate. Pseudorabies virus mutants differ in their interaction with heparin/heparan sulfate when altered for specific glycoprotein C heparin-binding domain

Cell surface heparan sulfate serves as an initial receptor for a number of herpesviruses including pseudorabies virus (PrV). It has been demonstrated that the heparan sulfate-binding domain of PrV glycoprotein C is composed of three discrete clusters of basic residues corresponding to amino acids 76-RRKPPR-81, 96-HGRKR-100, and 133-RFYRRGRFR-141, respectively, and that these clusters are functionally redundant, i.e. each of them could independently support PrV attachment to cells (Flynn, S. J., and Ryan, P. (1996) J. Virol. 70, 1355-1364). To evaluate the functional significance of each of these clusters we have used PrV mutants in which, owing to specific alterations in glycoprotein C, the heparan sulfate-binding site is dominated by a single specific cluster. These mutants exhibited different patterns of susceptibility to selectively N-, 2-O-, and 6-O-desulfated heparin preparations in virus attachment/infectivity assay. Moreover PrV mutants differed as regard to efficiency of their attachment to and infection of cells pretreated with relatively low amounts of heparan sulfate-degrading enzymes. Furthermore glycoprotein C species, purified from respective mutants, bound heparin oligosaccharide fragments of different minimum size. These differences suggest that specific clusters of basic amino acids of the heparan sulfate-binding domain of glycoprotein C may support PrV binding to different structural features/stretches within the heparan sulfate chain.     

The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform-specific

We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-betas (TGF-beta) with heparin and heparan sulfate. TGF-beta1 and -beta2, but not -beta3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-beta1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-beta1 by alpha2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-beta2.alpha2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-beta3 cannot be affected. Comparison of the amino acid sequences of the TGF-beta isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-beta dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain.     

Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin.     

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.     

Glycosaminoglycan binding properties of annexin IV, V, and VI

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.     

Differential binding of chemokines to glycosaminoglycan subpopulations

BACKGROUND: Specificity in leukocyte trafficking is likely to depend on sequential interactions between various cell-type-specific leukocyte adhesion molecules, such as selectins and integrin ligands, and leukocyte-activating factors. A major class of leukocyte-activating factors, the chemokines, are soluble polypeptides that bind glycosaminoglycans, the polysaccharide components of cell-surface and extracellular-matrix proteoglycans. It has been suggested that cell-surface glycosaminoglycans of the heparin/heparan sulfate class mediate the presentation of chemokines to leukocytes by vascular endothelial cells. We investigated the possibility that specificity exists in the recognition of particular heparin/heparan sulfate structures by chemokines, by studying the binding of four members of the chemokine superfamily to heparin and heparan sulfate. RESULTS: Using affinity co-electrophoresis we found that interleukin-8 preferentially bound a subfraction of heparin that also showed increased affinity for melanoma growth stimulating activity (also known as MGSA, GRO or GRO alpha). This same subfraction of heparin, however, was not significantly preferentially bound by platelet factor 4 or neutrophil activating factor-2. Subsequent analysis of the three-dimensional structures of these chemokines indicated that their ability to discriminate among heparin subspecies correlates with the presence of paired glutamic acid residues within the putative glycosaminoglycan-binding site of the chemokine. This observation led to predictions about the relative affinities of heparan sulfate for interleukin-8 and platelet factor 4, predictions that were confirmed by further binding assays. CONCLUSION: Chemokines can bind selectively to subsets of heparin/heparan sulfate glycosaminoglycans, raising the possibility that glycosaminoglycans participate in determining the specificity of leukocyte recruitment in vivo.     

Molecular characterization of Xenopus embryo heparan sulfate. Differential structural requirements for the specific binding to basic fibroblast growth factor and follistatin

Enzymatic elimination of heparan sulfate (HS) causes abnormal mesodermal and neural formation in Xenopus embryos, and HS plays an indispensable role in establishing the embryogenesis and tissue morphogenesis during early Xenopus development (Furuya, S., Sera, M., Tohno-oka, R., Sugahara, K., Shiokawa, K., and Hirabayashi, Y. (1995) Dev. Growth Differ. 37, 337-346). In this study, HS was purified from Xenopus embryos to investigate its disaccharide composition and binding ability to basic fibroblast growth factor (bFGF) and follistatin (FS), the latter being provided in two isoforms with core sequences of 315 and 288 amino acids (designated FS-315 and FS-288) originating from alternative mRNA splicing. Disaccharide composition analysis of the purified Xenopus HS showed the preponderance of a disulfated disaccharide unit with uronic acid 2-O-sulfate and glucosamine 2-N-sulfate, which has been implicated in the interactions with bFGF. Specific binding of the HS to bFGF and FS-288, the COOH-terminal truncated form, was observed in the filter binding assay, whereas HS did not bind to FS-315, indicating that the acidic Glu-rich domain of FS-315 precluded the binding. The binding of the HS to bFGF or FS-288 was markedly inhibited by heparin (HP) and various HS preparations, but not by chondroitin sulfate, supporting the binding specificity of HS. The binding specificity was further investigated using FS-288 and bovine intestinal [3H]HS. Competitive inhibition assays of the HS binding to FS-288 using size-defined HP oligosaccharides revealed that the minimum size required for significant inhibition was a dodecasaccharide, which is larger than the pentasaccharide required for bFGF binding. The binding affinity of FS to HS increased in the presence of activin, a growth/differentiation factor, which could be inactivated by direct binding to FS. These results, taken together, indicate that the structural requirement for binding of HS to bFGF and FS is different. HS may undergo dynamic changes in its structure during early Xenopus embryogenesis in response to the temporal and spatial expression of various growth/differentiation factors.     

Molecular features of the collagen V heparin binding site

A heparin binding region is known to be present within the triple helical part of the alpha1(V) chain. Here we show that a recombinant alpha1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual alpha1(V) chains and HepV are eluted at identical NaCl concentrations (0.35 M) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of alpha1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the alpha1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.     

Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma

Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.     

Hypereosinophilic syndrome in a child mosaic for a congenital triplication of the short arm of chromosome 8

Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.     

Binding of Sindbis virus to cell surface heparan sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.     

Deleted in colorectal carcinoma (DCC) binds heparin via its fifth fibronectin type III domain

DCC (deleted in colorectal carcinoma) is a broadly expressed cell-surface receptor. Netrin-1 was recently identified as a DCC ligand in brain, but the possibility of other DCC ligands was suggested by the finding that an anti-DCC antibody (clone AF5) neutralized netrin-1-dependent commissural axon outgrowth without blocking DCC/netrin-1 interactions. Here we have searched for alternative cell-surface DCC ligands. A DCC-Ig fusion protein bound to neural and epithelial derived cell lines, indicating that these lines express ligand(s) for DCC. The cell-surface binding activity was mediated by the loop between beta-strands F and G of the fifth fibronectin type III repeat FNIII-D5. The loop included the sequence KNRR, which resembles heparin-binding motifs in other proteins. Heparinase and heparitinase treatment of cells reduced binding of DCC-Ig, suggesting that heparan sulfate proteoglycans are cell-surface DCC ligand(s). This was further supported by heparin blocking experiments and by binding of DCC-Ig to immobilized heparan sulfate. The interaction between DCC-Ig and heparan sulfate/heparin, both on the surface of cells and immobilized on plastic, was blocked by the same anti-DCC antibody that blocks netrin-1-dependent commissural axon outgrowth. Taken together, these findings suggest that the DCC-Ig/heparin interaction may contribute to the biological activity of DCC.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20-40% of radioactivity in [35S]heparin. Fibronectin attached directly to Sepharose also bound [35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit. The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfate heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads. It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.