Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca2+ and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca2+ dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a Kd of approximately 21 nM for the interaction of annexin V with end-chain immobilized heparin and a Kd of approximately 49 nM for the interaction with end-chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The Kd of a heparin octasaccharide binding to annexin V was approximately 1 microM with a binding stoichiometry of 1:1.     

Characterization of annexin A1 glycan binding reveals binding to highly sulfated glycans with preference for highly sulfated heparan sulfate and heparin

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

The amino-terminal part of PRELP binds to heparin and heparan sulfate

PRELP (proline, arginine-rich end leucine-rich repeat protein) is an extracellular matrix leucine-rich repeat protein. The amino-terminal region of PRELP differs from that of other leucine-rich repeat proteins in containing a high number of proline and arginine residues. The clustered proline and basic residues are conserved in rat, bovine, and human PRELP. Although the function of PRELP is not yet known, the clustered arginine residues suggest a heparan sulfate/heparin-binding capacity. We show here that PRELP indeed binds heparin and heparan sulfate. Truncated PRELP without the amino-terminal region does not bind heparin. The dissociation constant for the interaction of PRELP with heparin was determined by an in solution binding assay and by surface plasmon resonance analysis to be in the range of 10-30 nm. A 6-mer heparin oligosaccharide was the smallest size showing binding to PRELP. The binding increased with increasing length up to an 18-mer and depended on the degree of sulfation of heparin as well as heparan sulfate. Sulfate groups at all positions were shown to be of importance for the binding. Fibroblasts bind PRELP, and this interaction is inhibited with heparin, suggesting a function for PRELP as a linker between the matrix and cell surface proteoglycans.     

Transglutaminase-2 interaction with heparin: identification of a heparin binding site that regulates cell adhesion to fibronectin-transglutaminase-2 matrix

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.     

Human skin chymotrypsin-like proteinase chymase. Subcellular localization to mast cell granules and interaction with heparin and other glycosaminoglycans

The subcellular localization of human skin chymase to mast cell granules was established by immunoelectron microscopy, and binding of chymase to the area of the dermo-epidermal junction, a basement membrane, was demonstrated immunocytochemically in cryosections incubated with purified proteinase prior to immunolabeling. Because heparin and heparan sulfate proteoglycans are major constituents of mast cell granules and basement membranes, respectively, the ability of chymase to bind to glycosaminoglycans (GAG) was investigated. Among a variety of GAGs, only binding of chymase to heparin and heparan sulfate appears physiologically significant. Binding was ionic strength-dependent, involved amino groups on the proteinase, and correlated with increasing GAG sulfate content, indicating a predominantly electrostatic association. Interaction with heparin was observed in solutions containing up to 0.5 M NaCl, and interaction with heparan sulfate was observed in solutions containing up to 0.3 M NaCl. Binding of heparin did not detectably affect catalysis of peptide substrates, but may reduce accessibility of proteinase to protein substrates. Measurements among a series of serine class proteinases indicated that heparin binding was a more common property of mast cell proteinases than proteinases stored in other secretory granules. Binding of chymase to heparin is likely to have a storage as well as a structural role within the mast cell granule, whereas binding of chymase to heparan sulfate may have physiological significance after degranulation.     

Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).     

Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate

The conservation of positively charged residues in the N terminus of the hepatitis C virus (HCV) envelope glycoprotein E2 suggests an interaction of the viral envelope with cell surface glycosaminoglycans. Using recombinant envelope glycoprotein E2 and virus-like particles as ligands for cellular binding, we demonstrate that cell surface heparan sulfate proteoglycans (HSPG) play an important role in mediating HCV envelope-target cell interaction. Heparin and liver-derived highly sulfated heparan sulfate but not other soluble glycosaminoglycans inhibited cellular binding and entry of virus-like particles in a dose-dependent manner. Degradation of cell surface heparan sulfate by pretreatment with heparinases resulted in a marked reduction of viral envelope protein binding. Surface plasmon resonance analysis demonstrated a high affinity interaction (KD 5.2 x 10-9 m) of E2 with heparin, a structural homologue of highly sulfated heparan sulfate. Deletion of E2 hypervariable region-1 reduced E2-heparin interaction suggesting that positively charged residues in the N-terminal E2 region play an important role in mediating E2-HSPG binding. In conclusion, our results demonstrate for the first time that cellular binding of HCV envelope requires E2-HSPG interaction. Docking of E2 to cellular HSPG may be the initial step in the interaction between HCV and the cell surface resulting in receptor-mediated entry and initiation of infection.     

Structural requirements for heparin/heparan sulfate binding to type V collagen

Collagen-proteoglycan interactions participate in the regulation of matrix assembly and in cell-matrix interactions. We reported previously that a fragment (Ile824-Pro950) of the collagen alpha1(V) chain, HepV, binds to heparin via a cluster of three major basic residues, Arg912, Arg918, and Arg921, and two additional residues, Lys905 and Arg909 (Delacoux, F., Fichard, A., Cogne, S., Garrone, R., and Ruggiero, F. (2000) J. Biol. Chem. 275, 29377-29382). Here, we further characterized the binding of HepV and collagen V to heparin and heparan sulfate by surface plasmon resonance assays. HepV bound to heparin and heparan sulfate with a similar affinity (KD approximately 18 and 36 nM, respectively) in a cation-dependent manner, and 2-O-sulfation of heparin was shown to be crucial for the binding. An octasaccharide of heparin and a decasaccharide of heparan sulfate were required for HepV binding. Studies with HepV mutants showed that the same basic residues were involved in the binding to heparin, to heparan sulfate, and to the cell surface. The contribution of Lys905 and Arg909 was found to be significant. The triple-helical peptide GPC(GPP)5G904-R918(GPP)5GPC-NH2 and native collagen V molecules formed much more stable complexes with heparin than HepV, and collagen V bound to heparin/heparan sulfate with a higher affinity (in the nanomolar range) than HepV. Heat and chemical denaturation strongly decreased the binding, indicating that the triple helix plays a major role in stabilizing the interaction with heparin. Collagen V and HepV may play different roles in cell-matrix interactions and in matrix assembly or remodeling mediated by their specific interactions with heparan sulfate.     

Interfacial basic cluster in annexin V couples phospholipid binding and trimer formation on membrane surfaces

Annexin V is an abundant eukaryotic protein that binds phospholipid membranes in a Ca(2+)-dependent manner. In the present studies, site-directed mutagenesis was combined with x-ray crystallography and solution liposome binding assays to probe the functional role of a cluster of interfacial basic residues in annexin V. Four mutants were investigated: R23E, K27E, R61E, and R149E. All four mutants exhibited a significant reduction in adsorption to phospholipid membranes relative to the wild-type protein, and the R23E mutation was the most deleterious. Crystal structures of wild-type and mutant proteins were similar except for local changes in salt bridges involving basic cluster residues. The combined data indicate that Arg(23) is a major determinant for interfacial phospholipid binding and participates in an intermolecular salt bridge that is key for trimer formation on the membrane surface. Together, crystallographic and solution data provide evidence that the interfacial basic cluster is a locus where trimerization is synergistically coupled to membrane phospholipid binding.     

Structural requirements for high-affinity heparin binding: alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase

An essential property of human extracellular superoxide dismutase (hEC-SOD) is its affinity for heparin and heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. The C-terminal domain of hEC-SOD plays the major role in this interaction. This domain has an unusually high content of charged amino acids: six arginine, three lysine, and five glutamic acid residues. In this study, we used alanine scanning mutagenesis of charged amino acids in the C-terminal domain to elucidate the requirements for the heparin/heparan sulfate interaction. As a tool in this study, we used a fusion protein comprising the C-terminal domain of hEC-SOD fused to human carbonic anhydrase II (HCAII). The interaction studies were performed using the surface plasmon resonance technique and heparin-Sepharose chromatography. Replacement of the glutamic acid residues by alanine resulted, in all cases, in tighter binding. All alanine substitutions of basic amino acid residues, except one (R205A), reduced heparin affinity. The arginine and lysine residues in the cluster of basic amino acid residues (residues 210-215), the RK-cluster, are of critical importance for the binding to heparin, and arginine residues promote stronger interactions than lysine residues.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

The fusion domain of HIV gp41 interacts specifically with heparan sulfate on the T-lymphocyte cell surface

Studies of the interaction of the 16 residue fusion peptide domain of human immunodeficiency virus glycoprotein gp41 (gp41(FD)) with T lymphocytes are outlined. Fluorescence measurements of changes in the electrostatic surface and dipole potentials of the plasma membrane following the interaction with gp41(FD) are described. The results show that gp41(FD) interacts with heparan sulfate located on the cell surface. This interaction is blocked by interleukin-8 and abolished by pre-treating the cells with heparitinase. The specificity of the reaction was also assessed by observations that soluble heparan sulfate competes with the cell membrane interaction whereas soluble heparin (at the levels utilized) does not. Following binding to heparan sulfate, the interaction with the membrane seems to take place in a cooperative manner with the formation of gp41(FD) trimers. In simpler phospholipid membranes, however, a trimeric complex does not appear to be the dominant mode of interaction. Finally, by repeating some of these studies within an imaging regime, it appears that the gp41(FD)-T-cell interaction takes place within specific domains on the cell surface to similarly localized heparan sulfate moieties.     

Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences

Numerous functions of heparan sulfate proteoglycans are mediated through interactions between their heparan sulfate glycosaminoglycan chains and extracellular ligands. Ligand binding specificity for some molecules, including many growth factors, is determined by complex heparan sulfate fine structure, where highly sulfated, iduronate-rich domains alternate with N-acetylated domains. Syndecan-4, a cell surface heparan sulfate proteoglycan, has a distinct role in cell adhesion, suggesting its chains may differ from those of other cell surface proteoglycans. To determine whether the specific role of syndecan-4 correlates with a distinct heparan sulfate structure, we have analyzed heparan sulfate chains from the different surface proteoglycans of a single fibroblast strain and compared their ability to bind the Hep II domain of fibronectin, a ligand known to promote focal adhesion formation through syndecan-4. Despite distinct molecular masses of glypican and syndecan glycosaminoglycans and minor differences in disaccharide composition and sulfation pattern, the overall proportion and distribution of sulfated regions and the affinity for the Hep II domain were similar. Therefore, adhesion regulation requires core protein determinants of syndecan-4.     

Heparan sulfate proteoglycans retain Noggin at the cell surface: a potential mechanism for shaping bone morphogenetic protein gradients.

Bone morphogenetic proteins (BMPs) are expressed broadly and regulate a diverse array of developmental events in vivo. Essential to many of these functions is the establishment of activity gradients of BMP, which provide positional information that influences cell fates. Secreted polypeptides, such as Noggin, bind BMPs and inhibit their function by preventing interaction with receptors on the cell surface. These BMP antagonists are assumed to be diffusible and therefore potentially important in the establishment of BMP activity gradients in vivo. Nothing is known, however, about the potential interactions between Noggin and components of the cell surface or extracellular matrix that might limit its diffusion. We have found that Noggin binds strongly to heparin in vitro, and to heparan sulfate proteoglycans on the surface of cultured cells. Noggin is detected only on the surface of cells that express heparan sulfate, can be specifically displaced from cells by heparin, and can be directly cross-linked to a cell surface proteoglycan in culture. Heparan sulfate-bound Noggin remains functional and can bind BMP4 at the plasma membrane. A Noggin mutant with a deletion in a putative heparin binding domain has reduced binding to heparin and does not bind to the cell surface but has preserved BMP binding and antagonist functions. Our results imply that interactions between Noggin and heparan sulfate proteoglycans in vivo regulate diffusion and therefore the formation of gradients of BMP activity.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

ZG16p, an animal homolog of β-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules

ZG16p is a soluble 16?kDa pancreatic protein having structural similarities with plant β-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.     

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.     

Nucleosomes bind to cell surface proteoglycans.

Material on the surface of activated T-cells was displaced following incubation with a sulfated polysaccharide, dextrin 2-sulfate (D2S), and purified by anion-exchange chromatography. This revealed a complex comprising histones H2A, H2B, H3, and H4 and DNA fragmented into 180-base pair units characteristic of mono-, di-, tri, and polynucleosomes, a pattern of fragmentation similar to that found in apoptotic cells. An antibody raised against the purified nucleosome preparation bound to the plasma membrane of activated T-cells confirming the surface location of nucleosomes. The interaction of sulfated polysaccharides with nucleosomes was investigated using a biotinylated derivative of D2S. It was found that sulfated polysaccharides bound to nucleosomes via the N termini of histones, especially H2A and H2B. Treatment of T-cells with either heparinase or heparitinase abolished nucleosome binding to plasma membranes. This suggests that nucleosomes are anchored to the surface of T-cells by heparan sulfate proteoglycans through an ionic interaction with the basic N-terminal residues in the histones. Furthermore, nucleosomes bound to the cell surface in this manner are then able to bind other sulfated polysaccharides, such as D2S, heparin, or dextran sulfate, through unoccupied histone N termini forming a complex comprising cell surface heparan sulfate proteoglycans, nucleosomes, and sulfated polysaccharides.     

Heparan sulfate-chondroitin sulfate hybrid proteoglycan of the cell surface and basement membrane of mouse mammary epithelial cells

Chondroitin sulfate represents approximately 15% of the 35SO4-labeled glycosaminoglycans carried by the proteoglycans of the cell surface and of the basolateral secretions of normal mouse mammary epithelial cells in culture. Evidence is provided that these chondroitin sulfate-carrying proteoglycans are hybrid proteoglycans, carrying both chondroitin sulfate and heparan sulfate chains. Complete N-desulfation but limited O-desulfation, by treatment with dimethyl sulfoxide, of the proteoglycans decreased the anionic charge of the chondroitin sulfate-carrying proteoglycans to a greater extent than it decreased the charge of their constituent chondroitin sulfate chains. Partial depolymerization of the heparan sulfate residues of the proteoglycans with nitrous acid or with heparin lyase also reduced the effective molecular radius of the chondroitin sulfate-carrying proteoglycans. The effect of heparin lyase on the chondroitin sulfate-carrying proteoglycans was prevented by treating the proteoglycan fractions with dimethyl sulfoxide, while the effect of nitrous acid on the dimethyl sulfoxide-treated proteoglycans was prevented by acetylation. This occurrence of heparan sulfate-chondroitin sulfate hybrid proteoglycans suggests that the substitution of core proteins by heparan sulfate or chondroitin sulfate chains may not solely be determined by the specific routing of these proteins through distinct chondroitin sulfate and heparan sulfate synthesizing mechanisms. Moreover, regional and temporal changes in pericellular glycosaminoglycan compositions might be due to variable postsynthetic modification of a single gene product.