Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias.

Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

PDGF suppresses the sulfation of CD44v and potentiates CD44v-mediated binding of colon carcinoma cells to fibrin under flow.

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen).

Franceschetti hereditary recurrent corneal erosion.

PURPOSE: To describe new affected individuals of Franceschetti's original pedigree of hereditary recurrent erosion and to classify a unique entity called Franceschetti corneal dystrophy. DESIGN: Observational case series. METHODS: Slit-lamp examination of 10 affected individuals was conducted. Biomicroscopic examinations were supplemented by peripheral corneal biopsy in 1 affected patient with corneal haze. Tissue was processed for light and electron microscopy and immunohistochemistry was performed. DNA analysis was carried out in 12 affected and 3 nonaffected family members. RESULTS: All affected individuals suffered from severe ocular pain in the first decade of life, attributable to recurrent corneal erosions. Six adult patients developed bilateral diffuse subepithelial opacifications in the central and paracentral cornea. The remaining 4 affected individuals had clear corneas in the pain-free stage of the disorder. Histologic and immunohistochemical examination of the peripheral cornea in a single patient showed a subepithelial, avascular pannus. There was negative staining with Congo red. DNA analysis excluded mutations in the transforming growth factor beta-induced (TGFBI) gene and in the tumor-associated calcium signal transducer 2 (TACSTD2) gene. CONCLUSION: We have extended the pedigree of Franceschetti corneal dystrophy and elaborated its natural history on the basis of clinical examinations. A distinctive feature is the appearance of subepithelial opacities in adult life, accompanied by a decreased frequency of recurrent erosion attacks. Its clinical features appear to distinguish it from most other forms of dominantly inherited recurrent corneal erosion reported in the literature.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome.

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome. Here we demonstrate a loss of C4S, as well as a reduction in C4ST-1 mRNA and protein expression, in primary fibroblasts from Costello syndrome patients. We go on to show that expression of oncogenic HRAS in normal fibroblasts can repress C4ST-1 expression, whereas interference with oncogenic HRAS signaling in Costello syndrome fibroblasts elevated C4ST-1 expression, thus identifying C4ST-1 as a negatively regulated target gene of HRAS signaling. Importantly, we show that forced expression of C4ST-1 in Costello fibroblasts could rescue the proliferation and elastogenesis defects associated with oncogenic HRAS signaling in these cells. Our results indicate reduced C4ST-1 expression and chondroitin sulfation imbalance mediating the effects of oncogenic HRAS signaling in the pathogenesis of Costello syndrome. Thus, our work identifies C4ST-1-dependent chondroitin sulfation as a downstream vulnerability in oncogenic RAS signaling, which might be pharmacologically exploited in future treatments of not only Costello syndrome and other RASopathies, but also human cancers associated with activating RAS mutations.

Chondroitin 4-O-sulfotransferase-1 regulates the chain length of chondroitin sulfate in co-operation with chondroitin N-acetylgalactosaminyltransferase-2.

Previously, we demonstrated that sog9 cells, a murine L cell mutant, are deficient in the expression of C4ST (chondroitin 4-O-sulfotransferase)-1 and that they synthesize fewer and shorter CS (chondroitin sulfate) chains. These results suggested that C4ST-1 regulates not only 4-O-sulfation of CS, but also the length and amount of CS chains; however, the mechanism remains unclear. In the present study, we have demonstrated that C4ST-1 regulates the chain length and amount of CS in co-operation with ChGn-2 (chondroitin N-acetylgalactosaminyltransferase 2). Overexpression of ChGn-2 increased the length and amount of CS chains in L cells, but not in sog9 mutant cells. Knockdown of ChGn-2 resulted in a decrease in the amount of CS in L cells in a manner proportional to ChGn-2 expression levels, whereas the introduction of mutated C4ST-1 or ChGn-2 lacking enzyme activity failed to increase the amount of CS. Furthermore, the non-reducing terminal 4-O-sulfation of N-acetylgalactosamine residues facilitated the elongation of CS chains by chondroitin polymerase consisting of chondroitin synthase-1 and chondroitin-polymerizing factor. Overall, these results suggest that the chain length of CS is regulated by C4ST-1 and ChGn-2 and that the enzymatic activities of these proteins play a critical role in CS elongation.

The chondroitin/dermatan sulfate synthesizing and modifying enzymes in laryngeal cancer: expressional and epigenetic studies.

BACKGROUND: Significant biochemical changes are observed in glycosaminoglycans in squamous cell laryngeal carcinoma. The most characteristics are in chondroitin/dermatan sulfate fine structure and proportion, which might be due to differential expression of the enzymes involved in their biosynthesis. The aim of the present work was the investigation in expressional and epigenetic level of the enzymes involved in chondroitin/dermatan sulfate biosynthesis in laryngeal cancer. METHODS: Tissues subjected to total RNA and DNA isolation, and protein extraction. The techniques used in this study were RT-PCR analysis, western blotting and methylation specific PCR. RESULTS: We identified that many enzymes were expressed in the cancerous specimens intensively. Dermatan sulfate epimerase was expressed exclusively in the cancerous parts and in minor amounts in healthy tissues; in the macroscopically normal samples it was not detected. Furthermore, chondroitin synthase I and chondroitin polymerizing factor were strongly expressed in the cancerous parts compared to the corresponding normal tissues. Sulfotransferases, like chondroitin 6 sulfotransferase 3, were highly expressed mainly in healthy specimens. CONCLUSIONS: The study of the various chondroitin/dermatan synthesizing enzymes revealed that they were differentially expressed in cancer, in human laryngeal cartilage, leading to specific chondroitin/dermatan structures which contributed to proteoglycan formation with specific features. The expression of the examined enzymes correlated with the glycosaminoglycan profile observed in previous studies.

Characterization and binding activity of the chondroitin/dermatan sulfate chain from Endocan, a soluble endothelial proteoglycan.

Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40 kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and are mostly clustered in tetra- (35%), hexa- (12%), and octa- (5%) saccharide domains. Highly sulfated D, E, and B disaccharide units (HexA2S-GalNAc6S, HexA-GalNAc4S6S, and HexA2S-GalNAc4S) were also detected in significant amounts in both chains and may account for the HGF/SF-binding activity of the CS/DS. This work establishes that HEK-293 cells can be engineered to provide a valuable source of Endocan with authentic CS/DS chains, enabling the purification of sufficient amounts for structural and/or binding analysis and providing a possible model of Endocan CS/DS chain organization.

Immunohistochemical level of unsulfated chondroitin disaccharides in the cancer stroma is an independent predictor of prostate cancer relapse.

The glycosaminoglycan chondroitin sulfate is significantly increased in the peritumoral stroma of prostate tumors compared with normal stroma and is an independent predictor of prostate-specific antigen (PSA) relapse following radical prostatectomy. In this study, we determined whether specific alterations in the sulfation pattern of glycosaminoglycan chains in clinically organ-confined prostate cancer are associated with PSA relapse. Immunoreactivity to distinct glycosaminoglycan disaccharide epitopes was assessed by manually scoring the staining intensity in prostate tissues from patients with benign prostatic hyperplasia (n = 19), early-stage cancer (cohort 1, n = 55 and cohort 2, n = 275), and advanced-stage cancer (n = 20). Alterations to glycosaminoglycans in benign and malignant prostate tissues were determined by cellulose acetate chromatography and high-pressure liquid chromatography. Glycosaminoglycan disaccharide epitopes were localized to the peritumoral stroma of clinically localized prostate cancer. The level of immunostaining for unsulfated disaccharides (C0S) in the peritumoral stroma, but not for 4-sulfated (C4S) or 6-sulfated disaccharides (C6S), was significantly associated with the rate of PSA relapse following radical prostatectomy. High levels of C0S immunostaining were determined to be an independent predictor of PSA relapse (1.6-fold, P = 0.020). Advanced-stage prostate cancer tissues exhibited reduced electrophoretic mobility for chondroitin sulfate and increased unsulfated disaccharides when compared with benign prostatic hyperplasia tissues, whereas the sulfated disaccharide levels were unaffected. The level of C0S immunostaining in the peritumoral stroma is an independent determinant of PSA failure in clinically localized prostate cancer. Specific alterations to chondroitin sulfate side chains occurring during tumor development may be a crucial step for disease progression in prostate cancer.

A role for a lithium-inhibited Golgi nucleotidase in skeletal development and sulfation.

Sulfation is an important biological process that modulates the function of numerous molecules. It is directly mediated by cytosolic and Golgi sulfotransferases, which use 3'-phosphoadenosine 5'-phosphosulfate to produce sulfated acceptors and 3'-phosphoadenosine 5'-phosphate (PAP). Here, we identify a Golgi-resident PAP 3'-phosphatase (gPAPP) and demonstrate that its activity is potently inhibited by lithium in vitro. The inactivation of gPAPP in mice led to neonatal lethality, lung abnormalities resembling atelectasis, and dwarfism characterized by aberrant cartilage morphology. The phenotypic similarities of gPAPP mutant mice to chondrodysplastic models harboring mutations within components of the sulfation pathway lead to the discovery of undersulfated chondroitin in the absence of functional enzyme. Additionally, we observed loss of gPAPP leads to perturbations in the levels of heparan sulfate species in lung tissue and whole embryos. Our data are consistent with a model that clearance of the nucleotide product of sulfotransferases within the Golgi plays an important role in glycosaminoglycan sulfation, provide a unique genetic basis for chondrodysplasia, and define a function for gPAPP in the formation of skeletal elements derived through endochondral ossification.

Increased expression of non-sulfated chondroitin correlates with adverse clinicopathological parameters in prostate cancer.

Chondroitin sulfate is a structurally diverse glycosaminoglycan, which contains a variable degree of sulfation that helps to determine its biological function. It is involved in the regulation of cellular activity and has been implicated in carcinogenesis. To determine if the non-sulfated chondroitin backbone has a functional role in prostate cancer, we analyzed its expression by immunohistochemistry using the 1B5 monoclonal antibody and a set of tissue microarrays constructed with 227 prostate specimen cores from 81 cases of benign prostate tissue and 77 cases of prostate cancer, of which 69 of these cases are matched. Non-sulfated chondroitin was found in the secretory epithelial cells and stromal regions of both prostatic adenocarcinoma and benign prostatic tissues, as well as in the basal cells of benign glands. A higher percentage of cancerous cells were stained positively for non-sulfated chondroitin as compared with benign secretory cells of the same patient. Cancerous cells stained more intensely for non-sulfated chondroitin. This increase in percentage of cells stained and increase in staining intensity were associated with higher pathological T stage and extraprostatic extension. Non-sulfated chondroitin expression (either staining intensity or percentage of cells stained) in adenocarcinoma and its peritumoral stroma correlated significantly with several clinicopathological parameters of unfavorable outcome, including higher pathological T stage and Gleason score, presence of tumor in both prostatic lobes, extraprostatic extension, seminal vesicle involvement and preoperative prostate-specific antigen levels. These data suggest that non-sulfated chondroitin is a potentially useful biomarker for prostate cancer, and may be involved in regulating prostate cancer behavior.

Alteration of free radical development by ophthalmic viscosurgical devices in phacoemulsification.

PURPOSE: To determine the effect of ophthalmic viscosurgical devices (OVDs) and irrigation/aspiration (I/A) settings on free radical development during phacoemulsification. SETTING: Department of Ophthalmology, Nippon Medical School, Tokyo, Japan. METHODS: Free radicals were detected by the electron spin resonance (ESR) spin-trap method. After a spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide 1%, was mixed with the irrigating solution, phacoemulsification simulation was performed in an eye model at 100% ultrasound power levels for 10, 20, and 30 seconds. A high I/A setting (35 mL/min with 250 mm Hg vacuum) and a low I/A setting (15 mL/min with 60 mm Hg vacuum) were used in combination with no OVD, sodium hyaluronate 1% (Healon), sodium hyaluronate 2.3% (Healon5), sodium hyaluronate 3%-chondroitin sulfate 4% (Viscoat), or sodium hyaluronate 1% (Opegan). Immediately after phacoemulsification, the solution in the anterior chamber was collected and signals from the spin adduct were measured using an ESR spectrometer. RESULTS: With the low setting, all OVDs significantly suppressed the signals for up to 20 seconds. At 30 seconds, the signals were still significantly smaller in all OVD groups, except Healon, than those in the no-OVD. With the high setting, the effect was not significant in the Healon and Opegan groups at 10 seconds; by 20 seconds and after, only Viscoat showed significant suppression. CONCLUSIONS: Free radical development in phacoemulsification can be altered by OVD properties and I/A settings. To reduce oxidative stress to the corneal endothelium, attention should be paid to the retention of OVDs in the anterior chamber during phacoemulsification.

Effects of low molecular weight heparin in obstructed kidneys: decrease of collagen, fibronectin and TGF-beta, and increase of chondroitin/dermatan sulfate proteoglycans and macrophage infiltration.

BACKGROUND: Heparin exerts beneficial effects in different experimental models of nephropathy, as observed by the preservation of the structural morphology of the kidney after heparin therapy. Here we investigate molecular and cellular events involved in the protective effects of heparin in the progression of renal disease after unilateral ureteral obstruction. METHODS: Thirty-six rats were divided into six groups: group C (control) was not subjected to any surgical manipulation; group S (sham) was subjected to surgical manipulation but without ureteral ligation; group UUO was subjected to ureteral obstruction and received no treatment; group UUO + S was subjected to ureteral obstruction and received saline subcutaneously (s.c.) once daily; group UUO + H was subjected to ureteral obstruction and received low molecular weight heparin (LMW-Hep; 4 mg/kg) s.c. once daily; and group C + H was not subjected to any surgical manipulation and received LMW-Hep (4 mg/kg) s.c. once daily. After 14 days, the content of collagen, fibronectin, total glycosaminoglycans (GAGS), chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs), transforming growth factor-beta (TGF-beta) and cellular infiltration were determined in the kidneys by immunohistochemical and biochemical techniques. RESULTS: Collagen, fibronectin, total GAGS, CS/DSPGs, TGF-beta and cellular infiltration increased significantly in group UUO. LMW-Hep treatment reduced collagen, fibronectin and TGF-beta, but induced an increase in the content of total GAGS, CS/DSPGs and macrophage infiltration in group UUO + H when compared with group UUO. CONCLUSIONS: LMW-Hep diminishes fibrosis in obstructed kidneys by downregulating the synthesis of collagen, fibronectin and TGF-beta. The mechanisms underlying the overproduction of CS/DSPGs and the increase in cellular infiltration upon LMW-Hep administration remain to be elucidated.

The TSG-6 and I alpha I interaction promotes a transesterification cleaving the protein-glycosaminoglycan-protein (PGP) cross-link.

During co-incubation of human inter-alpha-inhibitor (IalphaI) and human tumor necrosis factor-stimulated gene 6 protein (TSG-6) SDS-stable interactions are formed between the two proteins. We have analyzed the products of this reaction and characterized the mechanism of complex formation. Following the incubation seven new bands not previously identified were apparent in SDS-PAGE. Three of these bands did not contain TSG-6, including heavy chain (HC)1.bikunin, HC2.bikunin, and free bikunin. In addition high molecular weight complexes composed of the same components as I alpha I, including HC1, HC2, and bikunin, were formed. The formation of these complexes was prevented by the addition of hyaluronan. The cross-links stabilizing these complexes displaying properties similar to the protein-glycosaminoglycan-protein (PGP) cross-link. The TSG-6-containing SDS-stable complexes were composed of HC1.TSG-6 or HC2.TSG-6 exclusively. Both glycosylated and non-glycosylated TSG-6 participated in the complex formation. The HC.TSG-6 cross-links were different from the PGP cross-link and were determined to be ester bonds between the alpha-carbonyl of the C-terminal Asp of the heavy chain and most likely a hydroxyl group containing the TSG-6 residue. The mechanism involved cleaving the PGP cross-link of I alpha I during a transesterification reaction. A TSG-6 hydroxyl group reacts with the ester bond between the alpha-carbonyl of the C-terminal Asp residues of HC1 or HC2 and carbon-6 of an internal N-acetylgalactosamine of the chondroitin-4-sulfate chain. An intermediate is formed resulting in a partitioning of the reaction between HC(1 or 2).TSG-6 complexes and transfer of HC(1 or 2) to the chondroitin via competing pathways.

CD44-chondroitin sulfate interactions mediate leukocyte rolling under physiological flow conditions.

CD44 on leukocytes binds to its glycosaminoglycan (GAG) ligand, hyaluronic acid, and mediates the rolling of leukocytes on vascular endothelial cells. We previously reported that the recombinant CD44 protein binds to other GAGs, including chondroitin sulfates (CS), although the physiological significance of this interaction has remained unclear. Here we report that the CD44 expressed on mouse lymphoma BW5147 cells supports cell binding to immobilized CS under static conditions and mediates cell rolling in CS-coated glass capillary tubes under shear stresses ranging from 0.5 to 1.5 dyn/cm(2), which is within the physiological range of forces in venules. Both interactions were completely inhibited by pretreating the cells with an anti-CD44 antibody or by pretreating the CS with chondroitinase ABC, but not hyaluronidase. To address the role of the CD44-CS interaction in vivo, we examined the tissue localization of the CS that interacts with CD44. Interestingly, a recombinant CD44 fusion protein bound to hepatic sinuosoidal endothelial cells where CS was also expressed, as assessed by immunohistochemistry. These findings support the involvement of the CD44-CS interaction in the primary adhesion of lymphocytes to endothelial cells and raise the possibility that this interaction plays a role in the capture of CD44-positive cells, such as activated T cells and certain tumor cells, by the hepatic sinusoidal vasculature.

Biological effects of group IIA secreted phosholipase A(2).

Group IIA secreted phospholipase A(2) (sPLA(2)-IIA) is the most abundant element in human tissues of a large family of low molecular weight phospholipases A(2), which shows properties different from those displayed by the cytosolic phospholipase A(2) involved in the release of arachidonic acid. sPLA(2)-IIA behaves as a ligand for a group of receptors inside the C-type multilectin mannose receptor family and also interacts with heparan sulfate proteoglycans such as glypican, the dermatan/chondroitin sulfate-rich decorin, and the chondroitin sulfate-rich versican, thus being able to internalize to specific compartments within the cell and producing biological responses. This review provides a short summary of the biological actions of sPLA(2)-IIA on intracellular signaling pathways.

Alterations in the content and composition of glycosaminoglycans in human laryngeal carcinoma.

Glycosaminoglycans in normal and cancerous human laryngeal cartilage were isolated and characterized by means of enzyme susceptibility and high performance liquid chromatography. The known mammalian glycosaminoglycans were identified in all samples but their content and composition varied between normal and malignant samples. Chondroitin/dermatan sulphate was the major glycosaminoglycan in all cases, but its relative proportion was decreased in malignant samples. Its sulphation pattern showed that in normal samples it was sulphated mainly at the C6 position of galactosamine, whereas in malignant samples it was sulphated mainly at C4. Dermatan sulphate, expressed as a result of the different digestion of samples with chondroitinases, was present in very small amounts in normal samples (2.7% of total sulphated glycosaminoglycans) but increased in proportion up to 27.7% in malignant samples. The content of oversulphated chondroitin/dermatan was increased twofold in malignant samples. The content of heparan sulphate was increased almost fivefold in malignant samples as compared to normal ones. The content of hyaluronan was increased in malignant samples 3.5-fold, amounting to up to 11.4% of total glycosaminoglycans. These dramatic changes in the content and composition of glycosaminoglycans seemed to be characteristic of the tumour and independent of its status.

Heparin and chondroitin sulfate inhibit adenine nucleotide hydrolysis in liver and kidney membrane enriched fractions.

The inhibition of adenine nucleotide hydrolysis by heparin and chondroitin sulfate (sulfated polysaccharides) was studied in membrane preparations from liver and kidney of adult rats. Hydrolysis was measured by the activity of NTPDase and 5'-nucleotidase. The inhibition of NTPDase by heparin was observed at three different pH values (6.0, 8.0 and 10.0). In liver, the maximal inhibition observed for ATP and ADP hydrolysis was about 80% at pH 8.0 and 70% at pH 6.0 and 10.0. Similarly to the effect observed in liver, heparin caused inhibition of ATP and ADP hydrolysis that reached a maximum of 70% in kidney (pH 8.0). Na(+), K(+) and Rb(+) changed the inhibitory potency of heparin, suggesting that its effects may be related to charge interaction. In addition to heparin, chondroitin sulfate also caused a dose-dependent inhibition in liver and kidney membranes. The maximal inhibition observed for ATP and ADP hydrolysis was about 60 and 50%, respectively. In addition, the hepatic and renal activity of 5'-nucleotidase was inhibited by heparin and chondroitin sulfate, except for kidney membranes where chondroitin sulfate did not alter AMP hydrolysis. On this basis, the findings indicate that glycosaminoglycans have a potential role as inhibitors of adenine nucleotide hydrolysis on the surface of liver and kidney cell membranes in vitro.

Molecular cloning and expression of a human chondroitin synthase.

We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.

Molecular cloning and expression of chondroitin 4-sulfotransferase.

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.