Heparan sulfate proteoglycans are involved in opiate receptor-mediated cell migration

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.     

Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.     

Phenotypic and functional maturation of dendritic cells mediated by heparan sulfate

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.     

A peptide found by phage display discriminates a specific structure of a trisaccharide in heparin

A number of recent studies have shown that heparan sulfate can control several important biological events on the cell surface through changes in sulfation pattern. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult. Heparin, which is primarily produced by mast cells, is closely approximated by the structural analog heparan sulfate. Screening of heparin-associating peptides using phage display and antithrombin-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. The peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and heparan sulfate but does not associate with other glycosaminoglycans. HappY recognizes three consecutive monosaccharide residues in heparin through its three arginine residues. HappY should be a useful probe to detect heparin and heparan sulfate in studies of glycobiology.     

Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

Purification and characterization of novel heparan sulfate proteoglycans produced by murine erythroleukemia cells in the growing phase

Murine erythroleukemia cells (Friend erythroleukemia cells of a C-10-6 line) synthesized sulfated glycosaminoglycans consisting mainly of heparan sulfate (more than 95%) with a small amount of chondroitin 4-sulfate. The heparan sulfate occurred as proteoglycans, of which the cell-associated component was separated into urea-insoluble (UI) and urea-soluble (US) fractions. The UI proteoglycan consisted of a single homogeneous molecular species with an estimated Mr of 360,000 (C(UI)PG), whereas the US component was composed of two subfractions: a homogeneous species with an Mr of 280,000 (C(US)PGI) and a mixture of compounds with Mr values of less than 80,000 (C(US)PGII), which were isolated in yields of about 110, 340, and 80 micrograms of hexuronate (HexUA), respectively, from 1.37 g of an acetone powder prepared from 5.7 x 10(9) cells in the logarithmic phase of growth. The proteoglycan released into the medium (12 liters) was a single homogeneous species with an Mr of 320,000 (MPG) which was purified in a yield of 500 micrograms of hexuronate. The major, cell-associated proteoglycan, C(US)PGI, had very high contents of serine and glycine, accounting for approximately 80% of the total amino acids. This proteoglycan as well as the other two large proteoglycans, C(UI)PG and MPG, were highly resistant to degradation by various proteinases. These three proteoglycans, C(UI)PG, C(US)PGI, and MPG, had heparan sulfates with estimated Mr values of 32,000, 27,000, and 30,000. On the other hand, the Mr of the smaller proteoglycan, C(UI)PGII, was not significantly different before and after beta-elimination, indicating that it contains only a small peptide, if any. The heparan sulfate of this proteoglycan consisted of smaller and heterogeneous molecular species with Mr values of 26,000, 20,000, and 4,000. Digestion of these heparan sulfates with heparitinase I plus II resulted in almost complete depolymerization and gave six unsaturated disaccharides, delta HexUA-GlcNAc, delta HexUA-Glc-NAc(6-SO4), delta HexUA-GlcNSO3, delta HexUA-GlcNSO3 (6-SO4), delta HexUA(2-SO4)-GlcNSO3, and delta HexUA(2-SO4)-GlcNSO3(6-SO4). The relative amounts of these disaccharides generated from the individual heparan sulfates showed that an average ratio of sulfate residues to repeating disaccharide units of the C(US)PGII-derived heparan sulfate (0.97) was significantly higher than those of the other three large proteoglycan-derived glycosaminoglycans (0.54-0.70).     

Transport of heparan sulfate into the nuclei of hepatocytes

Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.     

PR-39 coordinates changes in vascular smooth muscle cell adhesive strength and locomotion by modulating cell surface heparan sulfate-matrix interactions

PR-39 is proline-rich peptide produced at sites of tissue injury. While the functional properties of this peptide have not been fully defined, PR-39 may be an important regulator of processes related to cell-matrix adhesion since it reportedly upregulates syndecan-4, which is a critical determinant of focal adhesion formation. The ability of PR-39 to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells (SMCs) in a fashion coordinated with syndecan-4 expression was investigated. Treatment of SMCs with PR-39 did not alter syndecan-1 mRNA, but did induce a two-fold increase in syndecan-4 mRNA (P < 0.0001) and significantly enhanced cell surface expression of both syndecan-4 (P < 0.01) and heparan sulfate (HS) (P < 0.05). These observations were consistent with an observed increase in cell-matrix adhesive strength (P < 0.05) and a reduction in cell speed (P < 0.01) on fibronectin-coated substrates. Incubation of PR-39 treated cells with a soluble fibronectin derived heparin-binding peptide, as a competitive inhibitor of heparan sulfate/matrix interactions, abolished these effects. These data suggest that PR-39 mediated alterations of cell adhesion and motility may be related, in part, to the increased expression of heparan sulfate glycosaminoglycans (GAGs) that accompany the upregulation of cell surface syndecan-4. Furthermore, this investigation supports the notion that factors which control syndecan-4 expression may play an important role in regulating adhesion related cell processes.     

Phage display mapping for peptide 11 sensitive sequences binding to laminin-1

We utilized a 9-mer random phage display library to identify sequences which bind to laminin-1 and elute with heparan sulfate or peptide 11 (CDPGYIGSR). Laminin-1 derivatized plates were used for biopanning. Three consecutive rounds of low pH elutions were carried out, followed by three rounds of specific elutions, each consisting of a heparan sulfate elution followed by a peptide 11 elution. The random sequence inserts were sequenced for phage populations eluted at low pH, by heparan sulfate and by peptide 11. Specifically eluted phage populations exhibited three classes of mimotopes for different regions in the cDNA derived amino acid sequence of the 67 kDa laminin binding protein (LBP). These regions were (1) a palindromic sequence known as peptide G, (2) a predicted helical domain corresponding to LBP residues 205-229, and (3) TEDWS-containing C-terminal repeats. All elution conditions also yielded phage with putative heparin binding sequences. We modeled the LBP(205-229) domain, which is strongly predicted to have a helical secondary structure, and determined that this region likely possesses heparin-binding characteristics located to one side of the helix, while the opposite side appears to contain a hydrophobic patch where peptide 11 could bind. Using ELISA plate assays, we demonstrated that peptide 11 and heparan sulfate individually bound to synthetic LBP(205-229) peptide. We also demonstrated that the QPATEDWSA peptide could inhibit tumor cell adhesion to laminin-1. These data support the proposal that the 67 kDa LBP can bind the beta-1 laminin chain at the peptide 11 region, and suggest that heparan sulfate is a likely alternate ligand for the binding interactions. Our results also confirm previous data suggesting that the most C-terminal region of the LBP, which contains the TEDWS repeats, is involved in cell adhesion to laminin-1, and we specifically implicate the repeat sequence in that activity.     

Glycosaminoglycans differentially bind HARP and modulate its biological activity

Heparin affin regulatory peptide (HARP) is a polypeptide belonging to a family of heparin binding growth/differentiation factors. The high affinity of HARP for heparin suggests that this secreted polypeptide should also bind to heparan sulfate proteoglycans derived from cell surface and extracellular matrix defined as extracellular compartments. Using Western blot analysis, we detected HARP bound to heparan sulfate proteoglycans in the extracellular compartments of MDA-MB 231 and MC 3T3-E1 as well as NIH3T3 cells overexpressing HARP protein. Heparitinase treatment of BEL cells inhibited HARP-induced cell proliferation, and the biological activity of HARP in this system was restored by the addition of heparin. We report that heparan sulfate, dermatan sulfate, and to a lesser extent, chondroitin sulfate A, displaced HARP bound to the extracellular compartment. Binding analyses with a biosensor showed that HARP bound heparin with fast association and dissociation kinetics (kass = 1.6 x 10(6) M-1 s-1; kdiss = 0.02 s-1), yielding a Kd value of 13 nM; the interaction between HARP and dermatan sulfate was characterized by slower association kinetics (kass = 0.68 x 10(6) M-1 s-1) and a lower affinity (Kd = 51 nM). Exogenous heparin, heparan sulfate, and dermatan sulfate potentiated the growth-stimulatory activity of HARP, suggesting that corresponding proteoglycans could be involved in the regulation of the mitogenic activity of HARP.     

Isolation and characterization of peptidoglycans in urine from patients with mucopolysaccharidoses

Urinary dermatan sulfate (DS) and heparan sulfate (HS) were purified from mucopolysaccharidosis patients. DS shows average mol. wt 8600-12,000 (approx. one half of tissue DS), iduronic acid content 99.1-99.6% (81.8% in tissue DS), core peptide mostly di- or tri-peptide (--Ser--Gly--or--Ser--Gly--Glu--). Molecular weight of HS ranged from 2500 to 20,000, averaging about 5000. Highly sulfated HS was found in the low molecular weight fraction, and no bound core peptide. By contrast, HS in the high molecular weight fraction bound one sulfate per repeating unit, which include core peptide.     

Blockade of alpha 5 beta 1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against alpha5beta1 integrins or by a peptide (GRGDSP) that binds to beta1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of beta1 or beta2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, alpha5beta1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by alpha5beta1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.     

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure delta UA2X(1----4)-D-GlcNY6X (where delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R.J. Linhardt, Anal. Biochem. 195, 68-73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of -2, delta UA2S(1----4)-D-GlcNAc and delta UA(1----4)-D-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

Fractionation and isolation of heparan sulfates using poly-L-lysine-Sepharose

A simple procedure for the isolation of heparan sulfates from pig lung using a poly-L-lysine-Sepharose column is described. Glycosaminoglycans are absorbed on poly-L-lysine-Sepharose at pH 7.5 and eluted with an NaCl linear gradient in the following order: hyaluronic acid (0.32 M NaCl), chondroitin (0.36 M NaCl), keratan sulfate (0.80 M NaCl), chondroitin 4-sulfate (0.86 M NaCl), chondroitin 6-sulfate (0.95 M NaCl), dermatan sulfate (0.91 M NaCl), heparan sulfate (1.2 M NaCl), and heparin (1.35 M NaCl). Based on these observations, isolation of heparan sulfate from pig lung crude heparan sulfate fractions which contain chondroitin sulfates and dermatan sulfate was attempted, using this chromatographic technique.     

Identification of cell-binding site of angiomodulin (AGM/TAF/Mac25) that interacts with heparan sulfates on cell surface.

Angiomodulin (AGM/TAF/mac25) is a 30-kDa glycoprotein that was identified as an integrin-independent cell adhesion protein secreted by human bladder carcinoma cells. AGM is highly accumulated in small blood vessels of tumor tissues. In the present study, we attempted to identify the cell surface receptor and the cell-binding site of AGM using ECV-304 human vascular endothelial cells and BALB/c3T3 mouse fibroblasts. Heparin, heparan sulfate, and dextran sulfate, but not chondroitin sulfate, inhibited both adhesion of the two cell lines to AGM-coated plates and binding of AGM to these cells. Treatment of cells with heparinase, but not chondroitinase, inhibited both cell adhesion to AGM and AGM binding to cells. These results strongly suggested that heparan sulfates are the major receptor for AGM. Furthermore, we determined a 20-amino acid sequence within AGM molecule as its major cell-binding site. The synthetic peptide for the cell-binding sequence showed cell adhesion activity comparable to that of AGM, and the activity was inhibited by heparin and heparan sulfate. The peptide competitively inhibited cell adhesion to AGM and the binding of AGM to cells. These results indicated that AGM binds to cells through interaction of the identified cell-binding sequence with heparan sulfates on cell surface. It was also found that the heparan sulfate-binding peptide inhibited the formation of capillary tube-like structures of vascular endothelial cells in culture.