Tandem mass spectrometry for characterization of unsaturated disaccharides from chondroitin sulfate,dermatan sulfate and hyaluronan

Fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from chondriotin sulfate, dermatan sulfate and hyaluronan. The positional isomers of the sulfate group of mono- and disulfated disaccharides were distinguished from each other by both positive- and negative-ion fast atom bombardment tandem mass spectra, which gave sufficient information characteristic of the isomers. The anomeric isomers of nonsulfated disaccharides were characterized by the technique in the positive-ion mode. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of trisulfated disaccharide.Abbreviations FABMS fast atom bombardment mass spectrometry - MI metastable ion - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - SIMS secondary ion mass spectrometry - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - GlcA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - UA-GalNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S-GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S-GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA2S-GalNAcDiS 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA-GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Pharmacological properties of dermatan sulfate, of a low molecular weight dermatan sulfate and of two oversulfated derivatives

The pharmacological properties of unfractionated dermatan sulfate (U-DS, mean MW 25 kd, range 12-45 kd) of a low molecular weight fraction (LMW-DS, mean MW 4 kd range 1.6-8 kd), and of 2 oversulfated derivatives (S-DS1 and S-DS2, 2 and 3.8 sulfate groups per disaccharide units) were investigated. In a purified system, LMW-DS, S-DS1 and S-DS2 were respectively 0.5, 10 and 17 times more potent than U-DS to catalyse thrombin inhibition by heparin cofactor II. Identical differences were observed for the respective anticoagulant activities (activated partial thromboplastin time and thrombin clotting time). After bolus IV injection of increasing doses the pharmacokinetic parameters of U-DS were slightly dose dependent, and the total clearance of LMW-DS was, on the average, 2 times higher. The patterns of disappearance of S-DS1 and S-DS2 were strongly dose dependent and became concave-convex, suggesting different mechanisms of clearance. After SC injection, the bioavailability was less than 50% for U-DS and at least 100% for LMW-DS. The antithrombotic activity (Wessler-thromboplastin model) of LMW-DS was 2 timer lower than that of U-DS. In contrast to their in vitro (and ex vivo) enhanced anticoagulant activities, the antithrombotic potency of S-DS1 was identical to that of U-DS, while, at the same doses S-DS2 was devoid of any activity.     

Conformational behavior of chondroitin and chondroitin sulfate in relation to their physical properties as inferred by molecular modeling

Chondroitin and chondroitin sulfates belong to the family of glycosaminoglycans. They are most widely distributed in animal tissues, where they are involved in structural functions and in cell-cell communication. Their basic structures consist of a disaccharidic repeating unit of beta-D-glucuronic acid (GlcA) and 2-acetamido-2-deoxy-beta-D-galactose (GalNAc), this latter being sulfated at different positions. Molecular mechanics has been applied to calculate the adiabatic energy maps for each of the constituting disaccharides of chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate using the MM3 force field. Based on these maps, higher levels of structural organization have been simulated. On one hand, the disordered state is studied through a Metropolis-based algorithm; the resulting chains present a behavior of semirigid polymers, with an order of stiffness: chondroitin 4-sulfate > chondroitin > chondroitin 6-sulfate. On the other hand, the exploration of the stable ordered forms leads to numerous helical conformations of comparable energies. Several of these conformations correspond to the experimentally observed ones. The ability of coordination with cations has also been explored, resulting in a preferential stereospecificity for calcium ions when compared to sodium ions.     

Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)

Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Deltadisaccharides including DeltaDiHA, DeltaDi0S, DeltaDi2S, DeltaDi4S, DeltaDi6S, DeltaDi2,4S, DeltaDi2,6S, DeltaDi4,6S, and DeltaDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4, 6S-galNAc.     

Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate

Recent glycobiology studies have suggested fundamental biological functions for chondroitin, chondroitin sulfate and dermatan sulfate, which are widely distributed as glycosaminoglycan sidechains of proteoglycans in the extracellular matrix and at cell surfaces. They have been implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development of the central nervous system. They also function as receptors for various pathogens. These functions are closely associated with the sulfation patterns of the glycosaminoglycan chains. Surprisingly, nonsulfated chondroitin is indispensable in the morphogenesis and cell division of Caenorhabditis elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of mutants of squashed vulva genes.     

Purification of heparin,dermatan sulfate and chondroitin sulfate from mixtures by sequential precipitation with various organic solvents

Heparin, dermatan sulfate and chondroitin sulfate in mixtures were fractionated by sequential precipitation with methanol, ethanol and propanol. The recovered fractions from 0.1 to 2.0 volumes of various solvents were analyzed by agarose-gel electrophoresis and densitometric analysis. Heparins with different relative percentages of slow-moving and fast-moving components were precipitated from 0.5 to 0.7 volumes of methanol, and in this range of volumes, the amount of slow-moving component of heparin decreases and that of the fast-moving species increases. From 0.8 to 1.6 volumes of methanol, mixtures with different percentages of the fast-moving component, dermatan sulfate and chondroitin sulfate are precipitated. Heparin was precipitated from mixtures in the range of 0.1 to 0.4 volumes of ethanol, and from 0.5 to 0.8 volumes mixtures with different relative percentages of dermatan sulfate and chondroitin sulfate were precipitated. From 1.0 to 2.0 volumes of ethanol, high purity (about 100%) chondroitin sulfate can be precipitated. Propanol induces the precipitation of heparin from 0.3 to 0.4 volumes, whilst dermatan sulfate with a purity greater than 85% is precipitated at 0.5 and 0.6 volumes of propanol. 100% chondroitin sulfate is obtained with volumes greater than 0.8. Heparin and chondroitin sulfate from a bovine lung extract of glycosaminoglycans were purified by sequential precipitation with ethanol. The fraction precipitated with 0.4 volumes of ethanol shows greater than 90% heparin and that recovered from 0.9 to 2.0 volumes is composed of 100% chondroitin sulfate.     

Binding of chondroitin sulfate, dermatan sulfate and fat-storing cell-derived proteoglycans to rat hepatocytes

1. The interaction of isolated rat hepatocytes with exogenous 3H-labeled chondroitin-4-sulfate and dermatan sulfate and with biosynthetically 35S-labeled proteoglycans secreted by cultured rat liver fat-storing cells has been studied. 2. All ligands are bound by hepatocytes in a concentration-dependent manner. Scatchard-plot analysis of the data revealed the existence of high- and low-affinity binding modes. 3. The cell-bound exogenous [3H]glycosaminoglycans could be displaced by each unlabeled ligand and by heparin, whereas displacement of the endogenous material was less effective. 4. Binding of all ligands to hepatocytes increased with time. For the exogenous glycosaminoglycans the two- to threefold amount was retained at 37 degrees C as compared to 4 degrees C; it was markedly reduced by pretreatment of the cells with trypsin. 5. Degradation of the exogenous ligands could be detected neither for the cell-bound fraction nor for the free glycosaminoglycans in the culture medium. 6. The binding of the ligands to hepatocytes is viewed as a cell-matrix interaction. Its possible pathobiochemical relevance in liver fibrosis or neoplasia is discussed.     

Conformational studies on five octasaccharides isolated from chondroitin sulfate using NMR spectroscopy and molecular modeling

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(beta1-3)GalNAc(beta1- disaccharide units, partly O-sulfated at different positions. The presence of the disulfated disaccharide D-unit, GlcA2S(beta1-3)GalNAc6S, in the CS moiety of the proteoglycan DSD-1-PG/phosphacan, correlates with neurite outgrowth promotion. The binding of monoclonal antibody (mAb) 473HD to DSD-1-PG, reducing neuronal stimulation, is inhibited by shark cartilage CS-D. CS-D is also recognized by two other mAbs, MO-225 and CS-56. Conformational studies were performed using NMR spectroscopy and molecular modeling on five octasaccharides isolated from shark cartilage CS-D. These octasaccharides present different binding properties toward the three mAbs. The combination of the experimental and theoretical approaches revealed that the sulfate group at position 2 of GlcA in disaccharide D and the presence of an exocyclic negative tail in disaccharides C [GlcA(beta1-3)GalNAc6S] and DeltaC [Delta4,5HexA(alpha1-3)GalNAc6S] are important for antibody recognition.     

Characterization of chondroitin sulfate and dermatan sulfate proteoglycans of extracellular matrices of human umbilical cord blood vessels and Wharton's jelly

The chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs) of the human umbilical cord vein, arteries and Wharton's jelly matrices were characterized and localized by immunohistochemical analysis. The CS/DSPGs were found to be decorins and biglycans with 43-48 kDa core proteins and are distributed throughout the umbilical cord. A truncated form of decorin having only the approximately 14 kDa NH(2)-terminal portion of the core protein was found exclusively in the vein. The proteoglycans, regardless of their locations, have two types of CS/DS chains, one with approximately 90% CS and approximately 10% DS and the other with approximately 65% CS and approximately 35% DS. The glycosaminoglycan (GAG) chains of the truncated decorin consist of approximately 53% CS and approximately 47% DS. Both decorin and biglycan including the truncated form of decorin could efficiently bind collagen I and fibronectin. The decorin and biglycan with approximately 10% DS and approximately 90% CS were loosely bound in the extracellular matrices, whereas those with approximately 35% DS bound strongly. Together, these data demonstrate that, the GAG chains with 35-47% DS but not those with 10% DS, interact strongly with the matrix. Our data also show that the GAG chain composition is a significant factor in binding of the decorin and biglycan to matrix proteins. The expression of decorin and biglycan with distinctively different CS/DS proportions implies specific biological functions for these PGs in the umbilical cord. The occurrence of the truncated form of decorin exclusively in the umbilical vein suggests a specific functional role.     

Study on the interaction of taiwaniaquinoids with FTO by spectroscopy and molecular modeling

In this work, an attempt has been made to study the interaction of four taiwaniaquinoids with fat mass and obesity-associated protein (FTO) by UV–vis absorption, fluorescence spectroscopy, and molecular docking techniques. The results indicated that taiwaniaquinoids effectively quenched the intrinsic fluorescence of FTO via static quenching. According to the binding constants and thermodynamic parameters at three different temperatures, the hydrophobic force and electrostatic interactions appeared be the predominant intermolecular forces in stabilizing the complex. Results revealed that W-4 was the strongest quencher and W-3 was the weakest. The results of synchronous and three-dimensional fluorescence spectra showed that the conformation of FTO was changed. In addition, the influence of molecular structure on the quenching effect has been investigated.     

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.     

Interaction of chondroitin sulfate and dermatan sulfate from various biological sources with heparin-binding growth factors and cytokines

Chondroitin sulfate (CS) and dermatan sulfate (DS) interact with various extracellular molecules such as growth factors, cytokines/chemokines, neurotrophic factors, morphogens, and viral proteins, thereby playing roles in a variety of biological processes including cell adhesion, proliferation, tissue morphogenesis, neurite outgrowth, infections, and inflammation/leukocyte trafficking. CS/DS are modified with sulfate groups at C-2 of uronic acid residues as well as C-4 and/or C-6 of N-acetyl-D-galactosamine residues, yielding enormous structural diversity, which enables the binding with numerous proteins. We have demonstrated that highly sulfated CS-E from squid cartilage, for example, interacts with heparin-binding proteins including midkine, pleiotrophin, and fibroblast growth factors expressed in brain with high affinity (Kd values in the nM range). Here, we analyzed the binding of CS and DS, which have a relatively low degree of sulfation and have been widely used as a nutraceutical and a drug for osteoarthritis etc., with a number of heparin-binding neurotrophic factors/cytokines using surface plasmon resonance (SPR) and structurally characterized the CS/DS chains. SPR showed that relatively low sulfated CS-A, DS, and CS-C also bound with significant affinity to midkine, pleiotrophin, hepatocyte growth factor, monokine-induced by interferon-γ, and stromal cell derived factor-1β, although the binding was less intense than that with highly sulfated CS-D and CS-E. These findings suggest that even low sulfated CS and/or DS chains may contain binding domains, which include fine sugar sequences with specific sulfation patterns, and that sugar sequences, conformations and electrostatic potential are more important than the simple degree of sulfation represented by disaccharide composition.     

The separation of chondroitin sulfate disaccharides and hyaluronan oligosaccharides by capillary zone electrophoresis

We have developed techniques for the separation of unsulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo- hex-4-enopyranosyluronicacid)-D-galactose and -D-glucose), monosulfated (2-acetamido-2-deoxy-3- O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-D-galactose and 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex- 4-enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose),disulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulfo-alpha-L-threo-hex-4- enopyranosyluronic acid)-4-sulfo-D-galactose and -6-sulfo-D-galactose and 2-acet-amido-2-deoxy-3-O-(4-deoxy-alpha-L-threo-hex-4-enopy- ranosyluronic acid)-4,6-di-O-sulfo-D-galactose), and trisulfated (2-acetamido-2-deoxy-3-O-(4-deoxy-2-O- sulfo-alpha-L-threo-hex-4-enopyranosyluronic acid)-4,6-di-O-sulfo-D-galactose) isomers of chondroitin using capillary zone electrophoresis. In addition, it is possible to separate oligomers of hyaluronan by similar protocols. These techniques represent a rapid, sensitive, and reproducible technique for the assay of these molecules from digests of connective tissues.     

Disialogangliosides and their interaction with cholera toxin - investigation by molecular modeling, molecular mechanics and molecular dynamics

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface disialogangliosides (GD1A, GD1B and GD3) in aqueous environment. The molecular mechanics calculation reveals that water mediated hydrogen bonding network plays a significant role in the structural stabilization of GD1A, GD1B and GD3. These water mediated hydrogen bonds not only exist between neighboring residues but also exist between residues that are separated by 2 to 3 residues in between. The conformational energy difference between different conformational states of gangliosides correlates very well with the number of water mediated and direct hydrogen bonds. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin is worked out. The NeuNAc has a limited allowed eulerian space at the binding site of Cholera Toxin (2.4%). The molecular modeling, molecular mechanics and molecular dynamics of disialoganglioside-cholera toxin complex reveal that cholera toxin can accommodate the disialoganglioside GD1A in three different modes. A single mode of binding is permissible for GD1B and GD3. Direct and water mediated hydrogen bonding interactions stabilizes these binding modes and play an essential role in defining the order of specificity for different disialogangliosides towards cholera toxin. This study not only provides models for the disialoganglioside-cholera toxin complexes but also identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Ultrasensitive capillary electrophoresis of sulfated disaccharides in chondroitin/dermatan sulfates by laser-induced fluorescence after derivatization with 2-aminoacridone

An ultrasensitive capillary electrophoretic method for separating the variously sulfated chondroitin/dermatan sulfate-derived Δ-disaccharides after digestion with chondro/dermatolyases and derivatization with the fluorophore 2-aminoacridone is described. All known mono-, di- and tri-sulfated Δ-disaccharides were completely separated using 15 mM orthophosphate buffer (pH 3.0) at 20 kV without any interference of the excess derivatizing reagent. They were detected at the anode (reversed polarity) using either an Ar-ion laser-induced fluorescence (LIF) detector (excitation wavelength 488 nm) or a UV detector. The sensitivity obtained by LIF (0.51 pmol/l) was at least 100 and 10 times higher as compared to those obtained by UV detection at 232 nm of underivatized Δ-disaccharides and at 254 nm of those derivatized with aminoacridone, respectively. The method has been easily applied to the analysis of chondroitin/dermatan sulfates from various tissues at the attogram level, including chondrotin/dermatan sulfates from normal and aneurysmal human abdominal aortas.     

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.     

Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44

Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.