Genetic analysis of proteoglycan structure, function and metabolism.

Significant progress has been made in understanding the structure, function, and metabolism of proteoglycans. Many of the advances derive from the application of recombinant DNA methodology to their core proteins and from the characterization of animal cell mutants altered in glycosaminoglycan synthesis.

Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate.

Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.

Identification of Heparan-Sulfate Rich Cells in the Loose Connective Tissues of the Axolotl (Ambystoma mexicanum) with the Potential to Mediate Growth Factor Signaling during Regeneration.

Limb regeneration is the outcome of a complex sequence of events that are mediated by interactions between cells derived from the tissues of the amputated stump. Early in regeneration, these interactions are mediated by growth factor/morphogen signaling associated with nerves and the wound epithelium. One shared property of these proregenerative signaling molecules is that their activity is dependent on interactions with sulfated glycosaminoglycans (GAGs), heparan sulfate proteoglycan (HSPG) in particular, in the extracellular matrix (ECM). We hypothesized that there are cells in the axolotl that synthesize specific HSPGs that control growth factor signaling in time and space. In this study we have identified a subpopulation of cells within the ECM of axolotl skin that express high levels of sulfated GAGs on their cell surface. These cells are dispersed in a grid-like pattern throughout the dermis as well as the loose connective tissues that surround the tissues of the limb. These cells alter their morphology during regeneration, and are candidates for being a subpopulation of connective tissue cells that function as the cells required for pattern-formation during regeneration. Given their high level of HSPG expression, their stellate morphology, and their distribution throughout the loose connective tissues, we refer to these as the positional information GRID (Groups that are Regenerative, Interspersed and Dendritic) cells. In addition, we have identified cells that stain for high levels of expression of sulfated GAGs in mouse limb connective tissue that could have an equivalent function to GRID cells in the axolotl. The identification of GRID cells may have important implications for work in the area of Regenerative Engineering.

A heparin-binding activity on Leishmania amastigotes which mediates adhesion to cellular proteoglycans.

The intracellular amastigote form of leishmania is responsible for the cell-to-cell spread of leishmania infection in the mammalian host. In this report, we identify a high-affinity, heparin-binding activity on the surface of the amastigote form of leishmania. Amastigotes of Leishmania amazonensis bound approximately 120,000 molecules of heparin per cell, with a Kd of 8.8 x 10(-8) M. This heparin-binding activity mediates the adhesion of amastigotes to mammalian cells via heparan sulfate proteoglycans, which are expressed on the surface of mammalian cells. Amastigotes bound efficiently to a variety of adherent cells which express cell-surface proteoglycans. Unlike wild-type CHO cells, which bound amastigotes avidly, CHO cells with genetic deficiencies in heparan sulfate proteoglycan biosynthesis or cells treated with heparitinase failed to bind amastigotes even at high parasite-input dosages. Cells which express normal levels of undersulfated heparan bound amastigotes nearly as efficiently as did wild-type cells. The adhesion of amastigotes to wild-type nonmyeloid cells was almost completely inhibited by the addition of micromolar amounts of soluble heparin or heparan sulfate but not by the addition of other sulfated polysaccharides.l Binding of amastigotes to macrophages, however, was inhibited by only 60% after pretreatment of amastigotes with heparin, suggesting that macrophages have an additional mechanism for recognizing amastigotes. These results suggest that leishmania amastigotes express a high-affinity, heparin-binding activity on their surface which can interact with heparan sulfate proteoglycans on mammalian cells. This interaction may represent an important first step in the invasion of host cells by amastigotes.

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata.

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan-deficient cells did not attach to substrate composed of a heparin-binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.

Thrombin adhesive properties: induction by plasmin and heparan sulfate.

We have previously demonstrated that chemically modified thrombin preparations induce endothelial cell (EC) adhesion, spreading and cytoskeletal reorganization via an Arg-Gly-Asp (RGD) sequence and the alpha v beta 3 integrin. Native thrombin, however, did not exhibit adhesive properties, consistent with crystal structure analysis, showing that Gly-Asp residues of the RGD epitope are buried within the molecule. We have now identified a possible physiological mean of converting thrombin to an adhesive protein. Plasmin, the major end product of the fibrinolytic system, converted thrombin to an adhesive protein for EC in a time and dose-dependent manner. EC adhesion and spreading was also induced by a low molecular weight (approximately 3,000 D) cleavage fragment generated upon incubation of thrombin with plasmin. Cell adhesion mediated by this fragment was completely inhibited by the synthetic peptide GRGDSP. Conversion of thrombin to an adhesive molecule was significantly enhanced in the presence of heparin or heparan sulfate, while other glycosaminoglycans (GAGs) (e.g., dermatan sulfate, keratan sulfate, chondroitin sulfate) had no effect. The role of cell surface heparan sulfate in thrombin conversion to EC adhesive protein was investigated using CHO cell mutants defective in various aspects of GAG synthesis. Incubation of both thrombin and a suboptimal amount of plasmin on the surface of formaldehyde fixed wild-type CHO-KI cells resulted in an efficient conversion of thrombin to an adhesive molecule, as indicated by subsequent induction of EC attachment. In contrast, there was no effect to incubation of thrombin and plasmin with fixed CHO mutant cells lacking both heparan sulfate and chondroitin sulfate, or with cells expressing no heparan sulfate and a three-fold increase in chondroitin sulfate. A similar gain of adhesive properties was obtained upon incubation of thrombin and plasmin in contact with native, but not heparinase-treated extracellular matrix (ECM) produced by cultured ECs. It appears that cell surface and ECM-associated heparan sulfate modulate thrombin adhesive properties through its heparin binding site in a manner that enables suboptimal amounts of plasmin to expose the RGD domain. Our results demonstrate, for the first time, a significant modulation of thrombin molecule by heparin, resulting in its conversion to a potent adhesive protein for ECs. This conversion is most effective in contact with cell surfaces, basement membranes and ECM.

Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans.

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.

Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes.

The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF-mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.

Killing of trypanosomes by the human haptoglobin-related protein.

African trypanosomes cause disease in humans and animals. Trypanosoma brucei brucei affects cattle but not humans because of its sensitivity to a subclass of human high density lipoproteins (HDLs) called trypanosome lytic factor (TLF). TLF contains two apolipoproteins that are sufficient to cause lysis of T. b. brucei in vitro. These proteins were identified as the human haptoglobin-related protein and paraoxonase-arylesterase. An antibody to haptoglobin inhibited TLF activity. TLF was shown to exhibit peroxidase activity and to be inhibited by catalase. These results suggest that TLF kills trypanosomes by oxidative damage initiated by its peroxidase activity.

Tumor formation dependent on proteoglycan biosynthesis.

The role proteoglycans play in tumor formation was examined by measuring the tumorigenicity of proteoglycan-deficient Chinese hamster ovary cell mutants in nude mice. When 10(7) cells were injected subcutaneously, mutants with less than about 15% of the wild-type level of proteoglycan synthesis did not produce tumors. Mutants defective in the synthesis of heparan sulfate proteoglycans also did not form tumors, whereas mutants with altered chondroitin sulfate proteoglycans were tumorigenic. Tumors arose from mixtures of wild-type and nontumorigenic mutant cells and contained both cell types, suggesting that wild-type cell proteoglycans enabled mutant cells to survive. The failure of heparan sulfate-deficient mutants to form tumors depended on the ability of the host to mount a B cell-mediated immune reaction.

Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.

Thrombospondin is a 420-kD platelet alpha-granule glycoprotein that binds specifically to heparin. We examined adhesion to thrombospondin of CHO K1 cells and three mutant CHO lines with varying deficiencies in glycosaminoglycan (GAG) synthesis. In an experiment in which the parent line (K1) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with less than 6% normal GAG synthesis had 11% adherence. CHO S677 cells, with decreased heparan sulfate proteoglycan but increased chondroitin sulfate proteoglycan, had 42% adherence. CHO S803 cells, with decreased heparan sulfate proteoglycan and normal chondroitin sulfate proteoglycan, had 31% adherence. Heparin inhibited K1 cell adhesion to thrombospondin, but not fibronectin, in a concentration-dependent manner. Dermatan sulfate but not chondroitin sulfate was also inhibitory. There was markedly decreased K1 cell adhesion to a thrombospondin core fragment that lacked the heparin binding NH2-terminal domain. Purified heparin binding domain, although poorly adhesive when adsorbed to substratum, inhibited cell adhesion to intact thrombospondin. Adhesion was better for all cell lines tested, including three human tumor cell lines, when thrombospondin was adsorbed at pH 4.0 compared with pH 7.4. When adsorption of thrombospondin was done at pH 7.4, cell adhesion was better when thrombospondin was adsorbed in the presence of greater than or equal to 0.6 mM calcium, compared to 0.1 mM calcium or EDTA. These findings suggest that thrombospondin can adsorb to plastic with varying degrees of exposure of a cell adhesion domain. We conclude that the thrombospondin cell adhesion receptor on CHO cells is a heparan sulfate proteoglycan, and that cell adhesion to thrombospondin depends on conformation of adsorbed thrombospondin.

Influence of core protein sequence on glycosaminoglycan assembly.

Recent studies have revealed a correlation between amino acid sequences around glycosylation sites in proteoglycans and the ability of cells to initiate and process glycosaminoglycan chains. Initiation depends on Ser-Gly/Ala dipeptides that have one or more acidic amino acids in close proximity. The formation of heparan sulfate chains depends on a nearby cluster of acidic residues, hydrophobic amino acids, and the close spacing of glycosylation sites.

Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycan chains from heparan sulfate proteoglycans present on the basolateral surface of hepatocytes and hepatoma cells in vitro. When injected into mice, CS protein is rapidly cleared from the blood circulation by hepatocytes. The binding region for the HSPGs is the evolutionarily conserved region II-plus of the CS protein. Here we have asked whether the presence of glycosaminoglycans on the plasma membrane of target cells is required for sporozoite invasion in vitro. Two types of target cells were used: HepG2 cells, which are permissive for Plasmodium berghei sporozoite development into mature exoerythrocytic forms, and CHO cells, in which the intracellular development of the parasites is arrested early after penetration. The invasion of mutant CHO cells expressing undersulfated glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion of CHO-K1 cells. Previous cleavage of HepG2 surface membrane glycosaminoglycans with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion and EEF development in these cells, although the glycosaminoglycan lyase treatments removed over 80% of CS binding sites from the cell surface. These results suggest that although the presence of glycosaminoglycans on the target cell surface enhances sporozoite invasion, glycosaminoglycans are not required for sporozoite penetration or the development of exoerythrocytic forms in vitro.

Reconstitution of the trypanolytic factor from components of a subspecies of human high-density lipoproteins.

Trypanosoma brucei brucei is non-infectious to man due to the sensitivity of these parasites to the lytic activity of normal human serum. Apolipoproteins (apo) have been purified, under non-denaturing conditions, from the subclass of human high-density lipoprotein (HDL), termed trypanosome lytic factor (TLF), which is responsible for the cytotoxicity of human serum to T. b. brucei. The TLF apolipoproteins were purified by anion exchange chromatography in the presence of the nonionic detergent octylglucoside and a reconstitution method was developed which allowed the role of the individual apolipoproteins and different lipids to be assessed. The results suggest that the TLF lipids do not have a direct role in lysis but are necessary for the correct assembly of the lytic HDL particle. Apo A-I, apo L-III and apo L-I contribute to lysis in reconstituted particles but individually they are not cytotoxic. Apo A-II was not required in the reconstituted TLF particle for trypanosome lysis. Formation of a lytic HDL particle required apo L-III suggesting its potential role as a toxin. Thermal inactivation of TLF activity correlated with the amount of denatured apo L-I, indicating that apo L-I was involved in lysis of T. b. brucei by native TLF.

Synthesis and glycosaminoglycan priming activity of three disaccharides related to the linkage region tetrasaccharide of proteoglycans.

To test if disaccharides might serve as primers of oligosaccharide synthesis in animal cells, we synthesized 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside, 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside, and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside. These three disaccharides are related to subunits of the linkage tetrasaccharide of heparan sulfate and chondroitin sulfate chains in animal cell proteoglycans. The disaccharides were synthesized with coupling efficiencies of 40-70% using thioglycosides or by activating the monosaccharides with trichloroacetimidate. The structures of these compounds were confirmed by 1H NMR, 13C NMR and elemental analysis. The ability of these disaccharides to prime glycosaminoglycan chains was examined in a Chinese hamster ovary cell mutant, p gsA 745, which lacks xylosyltransferase. The missing enzyme renders the cells dependent on exogenous primers for making glycosaminoglycan chains. 2-Naphthyl O-(beta-D-galactopyranosyl)-(1 --> 3)-beta-D-galactopyranoside and 2-naphthyl O-(beta-D-glucopyranosyluronic acid)-(1 --> 3)-beta-D-galactopyranoside did not stimulate glycosaminoglycan synthesis, but 2-naphthyl O-(beta-D-galactopyranosyl)-(1 --> 4)-beta-D-xylopyranoside at high concentration primed chains. The peracetylated derivative (2-naphthyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1 --> 4)- 2,3-di-O-acetyl-beta-D-xylopyranoside) primed chains at lower concentration (100 microM), suggesting that cells took up the compound and removed the acetyl groups apparently in the compartment where glycosaminoglycan synthesis occurs.

Synthesis and glycan priming activity of acetylated disaccharides.

Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-( 1 --> 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranoside (6), 2-naphthylmethyl 2-aceta-mido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2,4,6-tri-O-acetyl-beta-D-galactopyranoside (14), 2-naph-thylmethyl2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1 --> 3)-2-acetamido-4,6-di- O-acetyl-2-deoxy-alpha-D-galactopyranoside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glu-copyranosyl-(1 --> 6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (27). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin.

Loss of function in heparan sulfate elongation genes EXT1 and EXT 2 results in improved nitric oxide bioavailability and endothelial function.

BACKGROUND: Heparanase is the major enzyme involved in degradation of endothelial heparan sulfates, which is associated with impaired endothelial nitric oxide synthesis. However, the effect of heparan sulfate chain length in relation to endothelial function and nitric oxide availability has never been investigated. We studied the effect of heterozygous mutations in heparan sulfate elongation genes EXT1 and EXT2 on endothelial function in vitro as well as in vivo. METHODS AND RESULT: Flow-mediated dilation, a marker of nitric oxide bioavailability, was studied in Ext1(+/-) and Ext2(+/-) mice versus controls (n=7 per group), as well as in human subjects with heterozygous loss of function mutations in EXT1 and EXT2 (n=13 hereditary multiple exostoses and n=13 controls). Endothelial function was measured in microvascular endothelial cells under laminar flow with or without siRNA targeting EXT1 or EXT2. Endothelial glycocalyx and maximal arteriolar dilatation were significantly altered in Ext1(+/-) and Ext2(+/-) mice compared to wild-type littermates (glycocalyx: wild-type 0.67±0.1 µm, Ext1(+/-) 0.28±0.1 µm and Ext2(+/-) 0.25±0.1 µm, P<0.01, maximal arteriolar dilation during reperfusion: wild-type 11.3±1.0%), Ext1(+/-) 15.2±1.4% and Ext2(+/-) 13.8±1.6% P<0.05). In humans, brachial artery flow-mediated dilation was significantly increased in hereditary multiple exostoses patients (hereditary multiple exostoses 8.1±0.8% versus control 5.6±0.7%, P<0.05). In line, silencing of microvascular endothelial cell EXT1 and EXT2 under flow led to significant upregulation of endothelial nitric oxide synthesis and phospho-endothelial nitric oxide synthesis protein expression. CONCLUSIONS: Our data implicate that heparan sulfate elongation genes EXT1 and EXT2 are involved in maintaining endothelial homeostasis, presumably via increased nitric oxide bioavailability.