Chondroitin sulfate proteoglycans (CSPGs) are upregulated in the central nervous system following injury. Chondroitin sulfate glycosaminoglycan (CS GAG) side chains substituted on this family of molecules contribute to the limited functional recovery following injury by restricting axonal growth and synaptic plasticity. In the current study, the effects of degrading CS GAGs with Chondroitinase ABC (Ch'ase ABC) in the injured spinal cords of adult cats were assessed. Three groups were evaluated for 5 months following T10 hemisections: lesion-only, lesion+control, and lesion+Ch'ase ABC. Intraspinal control and Ch'ase ABC treatments to the lesion site began immediately after injury and continued every other day, for a total of 15 treatments, using an injectable port system. Delivery and in vivo cleavage were verified anatomically in a subset of cats across the treatment period. Recovery of skilled locomotion (ladder, peg, and beam) was significantly accelerated, on average, by >3 weeks in Ch'ase ABC-treated cats compared to controls. Ch'ase ABC-treated cats also showed greater recovery of specific skilled locomotor features including intralimb movement patterns and significantly greater paw placement onto pegs. Although recovery of basic locomotion (bipedal treadmill and overground) was not accelerated, intralimb movement patterns were more normal in the Ch'ase ABC-treated cats. Qualitative assessment of serotonergic immunoreactivity also suggested that Ch'ase ABC treatment enhanced plasticity. Finally, analyses using fluorophore-assisted carbohydrate electrophoresis (FACE) indicate CS GAG content is similar in cat and human. These findings show, for the first time, that intraspinal cleavage of CS GAGs can enhance recovery of function following spinal cord injury in large animals with sophisticated motor behaviors and axonal growth requirements similar to those encountered in humans.

Myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs) contribute to failed regeneration after neuronal injury. MAIs and CSPGs stimulate intracellular signals including the activation of RhoA and Rho kinase to block axonal extension through targeted modifications to the cytoskeleton. RhoA and ROCK are promising targets for therapeutic intervention to promote CNS repair; however, their ubiquitous expression will limit the specificity of drugs targeted to these molecules. We have identified the cytosolic phosphoprotein CRMP4b (collapsin-response mediator protein 4b) as a protein that physically and functionally interacts with RhoA to mediate neurite outgrowth inhibition. Short interfering RNA-mediated knockdown of CRMP4 promotes neurite outgrowth on myelin substrates, indicating a critical role for CRMP4 in neurite outgrowth inhibition. Disruption of CRMP4b-RhoA binding with a competitive inhibitor attenuates neurite outgrowth inhibition on myelin and aggrecan substrates. Stimulation of neuronal growth cones with Nogo leads to colocalization of CRMP4b and RhoA at discrete regions within the actin-rich central and peripheral domains of the growth cone, indicative of a potential function in cytoskeletal rearrangements during neurite outgrowth inhibition. Together, these data indicate that a RhoA-CRMP4b complex forms in response to inhibitory challenges in the growth cone environment and regulates cytoskeletal dynamics at distinct sites necessary for axon outgrowth inhibition. Competitive inhibition of CRMP4b-RhoA binding suggests a novel, highly specific therapeutic avenue for promoting regeneration after CNS injury.

The dystrophin-associated protein complex (DAPC) is necessary for maintaining the integrity of the muscle cell plasma membrane and may also play a role in coordinating signaling events at the cell surface. The alpha-/beta-dystroglycan subcomplex of the DAPC forms a critical link between the cytoskeleton and the extracellular matrix. A ligand blot overlay assay was used to search for novel dystroglycan binding partners in postsynaptic membranes from Torpedo electric organ. An approximately 125-kD dystroglycan-binding polypeptide was purified and shown by peptide microsequencing to be the Torpedo ortholog of the small leucine-rich repeat chondroitin sulfate proteoglycan biglycan. Biglycan binding to alpha-dystroglycan was confirmed by coimmunoprecipitation with both native and recombinant alpha-dystroglycan. The biglycan binding site was mapped to the COOH-terminal third of alpha-dystroglycan. Glycosylation of alpha-dystroglycan is not necessary for this interaction, but binding is dependent upon the chondroitin sulfate side chains of biglycan. In muscle, biglycan is detected at both synaptic and nonsynaptic regions. Finally, biglycan expression is elevated in muscle from the dystrophic mdx mouse. These findings reveal a novel binding partner for alpha-dystroglycan and demonstrate a novel avenue for interaction of the DAPC and the extracellular matrix. These results also raise the possibility of a role for biglycan in the pathogenesis, and perhaps the treatment, of muscular dystrophy.

The administration of large amounts of vitamin A to rabbits has been shown to result in depletion of cartilage matrix. The normal basophilic, metachromatic, and Alcian blue staining properties of the matrix are lost, especially in articular and epiphyseal cartilage. The cartilage cells remain intact, but are reduced in size. These changes sometimes appeared as early as 48 hours after the initiation of daily injection of 1 million units of vitamin A, and were usually well established by 5 days. Some rabbits failed to show changes in cartilage, even after 5 daily injections. Increased amounts of material presumed to be chondroitin sulfate were present in the sera of vitamin A-treated rabbits, usually by 72 hours after the first injection. This was demonstrated by a turbidimetric procedure using hexamminecobaltic chloride. In rabbits given sulfur-35 (Na(2)S(35)O(4)) 5 days before the initiation of vitamin A treatment, it was shown that sulfur-35 was lost from articular and epiphyseal cartilage. This was associated with an increase in the non-dialyzable sulfur-35 in both serum and in the cobalt-precipitable material. These rabbits also excreted more sulfur-35 than rabbits not given vitamin A. There was a reduction in sulfur-35 activity in chondromucoprotein extracted from the ear cartilage of vitamin A-treated rabbits. The changes are interpreted as indicating that the administration of large amounts of vitamin A to rabbits results in removal of chondroitin sulfate from cartilage matrix. The administration of small amounts of crude papain causes histologic changes in cartilage that are remarkably similar to those seen in vitamin A-treated rabbits. The possibility is suggested that the changes in cartilage produced by administration of vitamin A to rabbits may be the result of activation of a proteolytic enzyme or enzymes, with properties similar to those of papain.

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

BACKGROUND

Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36.

RESULTS

In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript.

CONCLUSIONS

Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci.

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

The adult brain contains a large population of oligodendrocyte precursor cells that can be identified using antibodies against the NG2 chondroitin sulfate proteoglycan. The functions of this newly recognized class of glial cells in the normal or pathological brain are not well understood. To begin to elucidate these functions, we have examined the morphology and distribution of oligodendrocyte precursor cells in the hippocampus and neocortex of normal and kainate-lesioned rats by anti-NG2 immunocytochemistry using light and electron microscopy. Large numbers of oligodendrocyte precursor cells were present in all layers of the neocortex and hippocampus. These cells differed in their morphology from astrocytes, oligodendrocytes and microglia. The processes of these cells often surrounded unlabeled areas of clear cytoplasm. At the electron microscopic level, some of the profiles that were enclosed by oligodendrocyte precursor cell processes contained synaptic vesicles. Other enclosed profiles were dendrites or dendritic spines. NG2-immunopositive processes were also observed to interpose between axon terminals containing round vesicles and dendrites with thick postsynaptic densities. After kainate injection, the NG2-positive oligodendrocyte precursor cells in the hippocampus displayed reactive changes characterized by swollen cell bodies, an increased number of small, filopodial-like processes, and higher levels of immunodetectable NG2. Both viable and degenerating oligodendrocyte precursor cells were observed with electron microscopy. These observations emphasize the dynamic nature of the oligodendrocyte precursor cell and suggest that, in addition to participating in the glial reactions to excitotoxic damage, oligodendrocyte precursor cells may regulate the stability, structure and function of synapses in the normal central nervous system.

Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma.

The binding of hyaluronate to SV-3T3 cells was measured by incubating a suspension of cells (released from the substratum with EDTA) with 3H-labeled hyaluronate and then applying the suspension to glass fiber filters which retained the cells and the bound hyaluronate. The extent of binding was a function of both the concentration of labeled hyaluronate and the cell number. Most of the binding took place within the first 2 min of the incubation and was not influenced by the presence or absence of divalent cations. The binding of labeled hyaluronate to SV-3T3 cells could be prevented by the addition of an excess of unlabeled hyaluronate. High molecular weight preparations of hyaluronate were more effective in preventing binding than low molecular weight preparations. The binding of [3H]hyaluronate was inhibited by high concentrations of oligosaccharide fragments of hyaluronate consisting of six sugars or more, and by chondroitin. The sulfated glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparin, and heparan sulfate) had little or no effect on the binding. The labeled hyaluronate bound to the cells could be totally removed by incubating the cells with testicular hyaluronidase, streptomyces hyaluronidase, or trypsin, indicating that the hyaluronate-binding sites are located on the cell surface.

Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self-association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin.

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.

Experimental therapeutics designed to enhance recovery from spinal cord injury (SCI) primarily focus on augmenting the growth of damaged axons by elevating their intrinsic growth potential and/or by nullifying the influence of inhibitory proteins present in the mature CNS. However, these strategies may also influence the wiring of intact pathways. The direct contribution of such effects to functional restoration after injury has been mooted, but as yet not been described. Here, we provide evidence to support the hypothesis that reorganization of intact spinal circuitry enhances function after SCI. Adult rats that underwent unilateral cervical spared-root lesion (rhizotomy of C5, C6, C8, and T1, sparing C7) exhibited profound sensory deficits for 4 weeks after injury. Delivery of a focal intraspinal injection of the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC (ChABC) was sufficient to restore sensory function after lesion. In vivo electrophysiological recordings confirm that behavioral recovery observed in ChABC-treated rats was consequent on reorganization of intact C7 primary afferent terminals and not regeneration of rhizotomized afferents back into the spinal cord within adjacent segments. These data confirm that intact spinal circuits have a profound influence on functional restoration after SCI. Furthermore, comprehensive understanding of these targets may lead to therapeutic interventions that can be spatially tailored to specific circuitry, thereby reducing unwanted maladaptive axon growth of distal pathways.

Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by approximately 70% following chondroitinase treatment, whereas binding of neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd approximately 1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd approximately 9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.

Perineuronal nets represent highly specialized glial and glia-associated structures. In this study, a triple fluorescence labeling of chondroitin sulfate proteoglycan-immunoreactive (CSPG-ir) and N-acetylgalactosamine (GalNac)-specific plant lectin Wisteria floribunda agglutinin (WFA) binding net components as well as parvalbumin-immunoreactivity (-ir) was performed. It was shown in the rat cortex, that the same nets frequently surrounding parvalbumin-ir neurons are stained by CSPG-ir as well as by the lectin binding method.

Glycosaminoglycans, a major component of the extracellular matrix molecules in animal tissues, play important roles in various physiological events. Glycosaminoglycans are found in not only vertebrates but also many invertebrates, implying a conserved function in the animal kingdom. Here, we discuss the analysis of glycosaminoglycans in 11 invertebrate phyla focusing on structure as well as physiological functions elucidated in model organisms. Various sulfated structures of heparan sulfate are widely distributed from very primitive organisms to humans, indicating an involvement in fundamental biological processes. By contrast, chondroitin/dermatan sulfate from lower organisms is limited in its structural complexity and often associated with a particular function. The presence of hyaluronic acid outside of vertebrates has been reported only in a mollusk.

Perlecan is a heparan sulfate proteoglycan and a major component of the glomerular basement membrane. To understand the role of heparan sulfate chains of perlecan in glomerular filtration, detailed analyses were performed of the kidneys of Hspg2(Delta)(3/)(Delta)(3) mice, whose perlecan lacks heparan sulfate attachment sites in N-terminal domain I. Macroscopic, histologic, and electron microscopic observations, as well as immunohistochemical and immunoelectron microscopic analyses using specific antibodies against perlecan and agrin core proteins, revealed no significant abnormalities in these mice under physiologic conditions. Polyethyleneimine staining demonstrated no significant changes in charge density in the glomerular basement membrane. Transcripts of other heparan sulfate proteoglycans, agrin, and collagen type XVIII, as well as perlecan, were expressed at similar levels to those in the wild-type littermates. Approximately 40% of the perlecan synthesized by Hspg2(Delta)(3/)(Delta)(3) fibroblasts was substituted with heparin sulfate and 60% was substituted with chondroitin sulfate. All of the perlecan synthesized by wild-type fibroblasts contained heparin sulfate, indicating an altered substitution of glycosaminoglycans on Hspg2(Delta)(3/)(Delta)(3) perlecan. Immunostaining indicated that the level of chondroitin sulfate was actually increased in the Hspg2(Delta)(3/)(Delta)(3) glomerular basement membrane. When administered intraperitoneally with BSA, Hspg2(Delta)(3/)(Delta)(3) mice exhibited remarkable proteinuria. These findings suggest that heparan sulfate chains of perlecan play an important role in glomerular filtration, especially of a large amount of protein.

The NG2 chondroitin sulfate proteoglycan is a membrane-associated molecule of approximately 500 kD with a core glycoprotein of 300 kD. Both the complete proteoglycan and a smaller quantity of the 300-kD core are immunoprecipitable with polyclonal and monoclonal antibodies against purified NG2. From some cell lines, the antibodies coprecipitate NG2 and type VI collagen, the latter appearing on SDS-PAGE as components of 140 and 250 kD under reducing conditions. The immunoprecipitation of type VI collagen does not seem to be due to recognition of the collagen by the antibodies, but rather to binding of the collagen to NG2. Studies on the NG2-type VI collagen complex suggest that binding between the two molecules is mediated by protein-protein interactions rather than by ionic interactions involving the glycosaminoglycans. Immunofluorescence double labeling in frozen sections of embryonic rat shows that NG2 and type VI collagen are colocalized in structures such as the intervertebral discs and arteries of the spinal column. In vitro the two molecules are highly colocalized on the surface of several cell lines. Treatment of these cells resulting in a change in the distribution of NG2 on the cell surface also causes a parallel change in type VI collagen distribution. Our results suggest that cell surface NG2 may mediate cellular interactions with the extracellular matrix by binding to type VI collagen.

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

The enzyme UDP-glucose dehydrogenase (Udpgdh) (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate, a critical component of the glycosaminoglycans, hyaluronan, chondroitin sulfate, and heparan sulfate. Although Udpgdh is a comparatively well characterized enzyme, no vertebrate genes encoding this enzyme have been reported to date. We report the cloning and characterization of the human and mouse UDP-glucose dehydrogenase genes. Mouse and human cDNAs predicted proteins of 493 and 494 amino acids, 24-25 residues longer at their carboxyl termini than the previously reported bovine Udpgdh sequence. The mouse Ugdh gene is composed of 10 exons, spanning 15 kilobases. Northern analyses indicated widespread expression of the gene in embryo and adult. Through interspecific backcross analyses, we localized the Ugdh gene to mouse chromosome 5 at approximately 39 centimorgans, suggesting that the human UGDH gene is localized to chromosome 4p13-15. Results from Southern analyses strongly suggest that Udpgdh is encoded by a single gene in the mouse. Transfection of mouse Ugdh expression vectors led to an increase in detectable Udpgdh activity in mammalian cells. Preliminary expression studies indicated that proinflammatory cytokines, such as interleukin 1beta, can substantially increase the expression of human UGDH in cultured human fibroblasts, suggesting that glycosaminoglycan biosynthesis may be partly regulated by the availability of activated UDP-glucuronate, as determined by relative Udpgdh expression levels.

We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an approximately 43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565-2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.

OBJECTIVE

Glycosaminoglycans (GAGs) contribute to the filtration barrier of aqueous outflow through the trabecular meshwork (TM). The purpose of this biochemical study was to identify the type and amount of GAGs in normal and in primary open-angle glaucoma (POAG) TM and adjacent anterior segment structures.

METHODS

The GAGs of 21 masked individual normal and POAG human TMs, as well as iris, ciliary body, and anterior sclera, were isolated biochemically, identified by selective GAG-degrading enzymes, and quantitated by computer-enhanced densitometry.

RESULTS

In 10 normal TMs (8 donors, 65 to 83 years of age), the GAG profile was: hyaluronic acid (0.77 +/- 0.26 ng/microgram dry-defatted weight +/- SEM); chondroitin 4(6-) sulfates and dermatan sulfate, collectively referred to as chondroitin sulfates (1.90 +/- 0.13 ng); keratan sulfates (0.33 +/- 0.06 ng); heparitin sulfates (2.02 +/- 0.52 ng); GAG enzyme-resistant material (0.02 +/- 0.01 ng); and total GAGs (5.05 +/- 0.70 ng). In 10 POAG TMs (6 donors, 67 to 88 years of age), the GAG profile was: hyaluronic acid (0.18 +/- 0.11 ng; P < 0.02, a 77% decrease; 6 of 10 TMs contained no detectable hyaluronic acid); chondroitin sulfates (2.39 +/- 0.31 ng); keratan sulfates (0.21 +/- 0.06 ng); heparitin sulfates (1.36 +/- 0.43 ng); GAG enzyme-resistant material (0.08 +/- 0.01 ng; P < 0.02); and total GAGs (4.09 +/- 0.33 ng; statistically insignificant). In the POAG iris, hyaluronic acid content was less (82% decrease, P < 0.02), and the chondroitin sulfates content was higher (72% increase, P < 0.02). Similarly, the POAG ciliary body and anterior sclera contained less hyaluronic acid and more chondroitin sulfates. The GAG profile of a "glaucoma suspect" donor specimen was similar to that of the POAG donor specimen.

CONCLUSIONS

The data provide the first quantitative biochemical profiles of GAGs of individual normal and POAG TM, and we suggest that a depletion of hyaluronic acid and the accumulation of chondroitin sulfates may increase aqueous outflow resistance in the POAG TM:

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Adhesion of urinary crystals to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones. The interaction between renal epithelial cells (BSC-1 line) and the most common crystal in kidney stones, calcium oxalate monohydrate (COM), was studied in a tissue culture model system. COM crystals bound to the cell surface within seconds in a concentration-dependent manner to a far greater extent than did brushite, another calcium-containing crystal found in urine. Adhesion of COM crystals to cells was blocked by the polyanion, heparin. Other glycosaminoglycans including chondroitin sulfate A or B, heparan sulfate, and hyaluronic acid, but not chondroitin sulfate C, prevented binding of COM crystals. Two nonsulfated polyanions, polyglutamic acid and polyaspartic acid, also blocked adherence of COM crystals. Three molecules found in urine, nephrocalcin, uropontin, and citrate, each inhibited binding of COM crystals, whereas Tamm-Horsfall glycoprotein (THP) did not. Prior exposure of crystals but not cells to inhibitory molecules blocked adhesion, suggesting that these agents exert their effect at the crystal surface. Inhibition of crystal binding followed a linear Langmuir adsorption isotherm for each inhibitor identified, suggesting that these molecules bind to a single class of sites on the crystal that are important for adhesion to the cell surface. Inhibition of crystal adhesion by heparin was rapidly overcome by the polycation protamine, suggesting that the glycosaminoglycan regulates cell-crystal interactions in a potentially reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)