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Bifunctional chondroitin synthase K4CP catalyzes glucuronic acid and N-acetylgalactosamine transfer activities and polymerizes a chondroitin chain. Here we have determined that an N-terminal region (residues 58-134) coordinates two transfer reactions and enables K4CP to catalyze polymerization. When residues 58-107 are deleted, K4CP loses polymerase activity while retaining both transfer activities. Peptide (113)DWPSDL(118) within this N-terminal region interacts with C-terminal peptide (677)YTWEKI(682). The deletion of either sequence abolishes glucuronic acid but not N-acetylgalactosamine transfer activity in K4CP. Both donor bindings and transfer activities are lost by mutating (677)YTWEKI(682) to (677)DAWEDI(682). On the other hand, acceptor substrates retain their binding to K4CP mutants. The characteristics of these K4CP mutants highlight different states of the enzyme reaction, providing an underlying structural basis for how these peptides play essential roles in coordinating the two glycosyltransferase activities for K4CP to elongate the chondroitin chain.

Chondroitin sulfate is a linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4–9) and temperature (30–60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

Secreted from intestine, human fibroblast growth factor 19 (hFGF19) is an endocrine metabolic regulator that controls bile acid synthesis in the liver. Earlier studies have suggested that hFGF19 at 10–100 nm levels signals through FGF receptor 4 (FGFR4) in the presence of a co-receptor, betaKlotho, but its activity and receptor specificity at physiological concentrations (picomolar levels) remain unclear. Here we report that hFGF19 at picomolar levels require sulfated glycosaminoglycans (sGAGs), such as heparan sulfate, heparin, and chondroitin sulfates, for its signaling via human FGFR4 in the presence of human betaKlotho. Importantly, sGAGs isolated from liver are highly active in enhancing the picomolar hFGF19 signaling. At nanomolar levels, in contrast, hFGF19 activates all types of human FGFRs, i.e. FGFR1c, FGFR2c, FGFR3c, and FGFR4 in the co-presence of betaKlotho and heparin and activates FGFR4 even in the absence of betaKlotho. These results show that sGAGs play crucial roles in specific and sensitive hFGF19 signaling via FGF receptors and suggest that hepatic sGAGs are involved in the highly potent and specific signaling of picomolar hFGF19 through FGFR4 and betaKlotho. The results further suggest that hFGF19 at pathological concentrations may evoke aberrant signaling through various FGF receptors.

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA beta1-4 and GlcNAc alpha1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153-164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st(f/f)) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1(f/f)) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st(f/f)AlbCre(+) mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1(f/f)AlbCre(+) mice. In contrast, Hs6st1(f/f)AlbCre(+) mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.

We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit (Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem.278 ,26780 -2678712740361). In the present study, we investigated the potential of other 3OST isoforms to produce Di-tetraS with heparan sulfate and heparin as acceptor substrates. 3OST-2, 3OST-3, and 3OST-4 produce Di-tetraS units as a major product from both substrates. 3OST-5 showed the same specificity for heparin, but the production from heparan sulfate was very low. Di-tetraS production by 3OST-1 was negligible. We then investigated the presence of Di-tetraS units in heparan sulfates from various rat tissues. Di-tetraS was detected in all of the tissues analyzed. Liver and spleen contain relatively high levels of Di-tetraS, 1.6 and 0.95%, respectively. However, the content of this unit in heart, large intestine, ileum, and lung is low, less than 0.2%. We further determined the expression levels of 3OST transcripts by quantitative real time PCR. The 3OST-3 transcripts are highly expressed in spleen and liver. The 3OST-2 and -4 are specifically expressed in brain. These results indicate that the Di-tetraS unit is widely distributed throughout the body as a rare and unique component of heparan sulfate and is synthesized by tissue-specific 3OST isoforms specific for Di-tetraS production.

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.

Contains fulltext : 53176.pdf (Publisher’s version ) (Open Access) Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin.

Bone morphogenetic protein (BMP)-2/4 play critical roles in early embryogenesis and skeletal development. BMP-2/4 signals conduct into cells via two types of serine/threonine kinase receptors, known as BMPR-I (IA and IB) and BMPR-II. Here we identified splicing factor 3b subunit 4 (SF3b4) as a molecule that interacts with BMPR-IA, using a yeast two-hybrid screening with a human fetal brain cDNA library. Co-immunoprecipitation/immunoblot analysis confirmed their interaction in mammalian cells. By separation of the cell components, SF3b4 was present in the cell membrane fraction with BMPR-IA as well as in the nucleus. Overexpression of SF3b4 inhibited BMP-2-mediated osteogenic and chondrocytic differentiation of C2C12 and ATDC5 cells, respectively, and the endogenous expression level of SF3b4 decreased during differentiation in ATDC5 cells. By reporter gene assay, SF3b4 suppressed Id reporter gene activity, specific to the Smad1/5/8 pathway, but not TGFbeta-mediated reporter gene activity. Biotin labeling of the cell surface proteins followed by their immunoblot revealed that SF3b4 decreased the cell surface BMPRI-A levels. Further analysis by molecular modeling of the intracellular domain of BMPR-IA, coupled with binding studies of its several mutants, indicated that the site(s) for SF3b4 binding is not directly associated with the C-terminal lobe and the activation segment. Taken together, these results suggest that SF3b4, known to be localized in the nucleus and involved in RNA splicing, binds BMPR-IA and specifically inhibits BMP-mediated osteochondral cell differentiation.

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

MY-174, a monoclonal antibody that reacts with specific sialylated O-linked glycoconjugates of chick SPACR (sialoprotein associated with cones and rods), also recognizes another molecule of 300 kDa. Here, we verified that this 300-kDa molecule is chick SPACRCAN (sialoproteoglycan associated with cones and rods), another member of a novel interphotoreceptor matrix molecule family. Screening for chick SPACRCAN was carried out by plaque hybridization using a probe for chick SPACR. Specific polyclonal antibodies raised against chick SPACRCAN were used for the following experiments. To determine whether the 300-kDa molecule detected by MY-174 was identical to 300-kDa chick SPACRCAN, the migrations of these bands were examined after various glycosidase digestions. Furthermore, the expression levels were measured during retinal development and compared with those of chick SPACR. The results demonstrated that the 300-kDa molecule recognized by MY-174 was chick SPACRCAN, and we further identified it as a proteoglycan with chondroitin sulfate chains. SPACRCAN had heavily sialylated N- and O-linked glycoconjugates, and its MY-174 antigenicity was abolished by O-glycanase treatment after neuraminidase treatment, as observed for chick SPACR. During retinal development, the mRNA and core protein expression levels, MY-174 antigenicity, and hyaluronan binding ability of SPACRCAN peaked around embryonic day 17 and then gradually decreased, whereas the corresponding expression levels of SPACR simply increased, but not its hyaluronan binding ability. The MY-174 reactivity of SPACRCAN in the adult retina was decreased compared with that in the newborn retina, whereas that of SPACR was increased. The decreased hyaluronan binding of SPACR was induced by an inhibitory effect of the excess of sialic acids in the adult stage. Thus, with similar core protein structures and specific sialylated glycoconjugates but distinct chondroitin sulfate chains, SPACRCAN and SPACR may have separate roles in the retina due to their differing expression profiles during development.

We have shown previously that a highly sulfated sequence, GalNAc(4,6-SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), is present at the nonreducing terminal of chondroitin sulfate (CS), and this structure was synthesized from a unique sequence, GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)), by sulfation with N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase. Uronosyl 2-O-sulfotrasferase (2OST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 2 of the GlcA residue of CS, is expected to be involved in synthesis of these structures; however, the specificity of 2OST concerning recognition of the sulfation pattern of the acceptor has largely remained unclear. In the present study, we examined the specificity of 2OST in terms of recognition of the sulfation pattern around the targeting GlcA residue. The recombinant 2OST could sulfate CS-A, CS-C, and desulfated dermatan sulfate. When [(35)S]glycosaminoglycans formed from CS-A after the reaction with the recombinant 2OST and [(35)S]PAPS were subjected to limited digestion with chondroitinase ACII, a radioactive tetrasaccharide (Tetra A) was obtained as a sole intermediate product. The sequence of Tetra A was found to be DeltaHexA-GalNAc(4SO(4))-GlcA(2SO(4))-GalNAc(6SO(4)) by enzymatic and chemical reactions. These observations indicate that 2OST transfers sulfate preferentially to the GlcA residue located in a unique sequence, -GalNAc(4SO(4))-GlcA-GalNAc(6SO(4))-. When oligosaccharides with different sulfation patterns were used as the acceptor, GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) and GlcA-GalNAc(4SO(4))-GlcA-GalNAc(6SO(4)) were the best acceptors for 2OST among trisaccharides and tetrasaccharides, respectively. These results suggest that 2OST may be involved in the synthesis of the highly sulfated structure found in CS-A.

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.

Specific inhibitors of hyaluronan (HA) biosynthesis can be valuable therapeutic agents to prevent cancer invasion and metastasis. We have found previously that 4-methylumbelliferone (MU) inhibits HA synthesis in human skin fibroblasts and in group C Streptococcus. In this paper, the inhibition mechanism in mammalian cells was investigated using rat 3Y1 fibroblasts stably expressing HA synthase (HAS) 2. Exposure of the transfectants to the inhibitor resulted in significant reduction of HA biosynthesis and matrix formation. The evaluation of HAS transcripts and analysis of cell-free HA synthesis demonstrated the post-transcriptional suppression of HAS activity by MU. Most interesting, the post-transcriptional suppression of HAS activity was also observed using p-nitrophenol, a well known substrate for UDP-glucuronyltransferases (UGT). We investigated whether the inhibition was exerted by the glucuronidation of MU using both high pressure liquid chromatography and TLC analyses. The production of MU-glucuronic acid (GlcUA) was consistent with the inhibition of HA synthesis in HAS transfectants. MU-GlcUA was also detected at a similar level in control cells, suggesting that the glucuronidation was mediated by an endogenous UGT. Elevated levels of UGT significantly enhanced the inhibitory effects of MU. In contrast, the inhibition by MU was diminished to the control level when an excess of UDP-GlcUA was added to the cell-free HA synthesis system. We propose a novel mechanism for the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UGT resulting in a depletion of UDP-GlcUA.

Hyaluronan (HA) is a large linear polymer of repeating disaccharides of glucuronic acid and GlcNAc. Although HA is widely distributed in vertebrate animals, it has not been found in invertebrates, including insect species. Insects utilize chitin, a repeating beta-1,4-linked homopolymer of GlcNAc, as a major component of their exoskeleton. Recent studies illustrate the similarities in the biosynthetic mechanisms of HA and chitin and suggest that HA synthase (HAS) and chitin synthase have evolved from a common ancestral molecule. Although the biochemical properties and in vivo functions of HAS proteins have been extensively studied, the molecular basis for HA biosynthesis is not completely understood. For example, it is currently not clear if proper chain elongation and secretion of HA require other components in addition to HAS. Here, we demonstrate that a non-HA-synthesizing animal, the fruit fly Drosophila melanogaster, can produce HA in vivo when a single HAS protein is introduced. Expression of the mouse HAS2 gene in Drosophila tissues by the Gal4/UAS (upstream activating sequence) system resulted in massive HA accumulation in the extracellular space and caused various morphological defects. These morphological abnormalities were ascribed to disordered cell-cell communications due to accumulation of HA rather than disruption of heparan sulfate synthesis. We also show that adult wings with HA can hold a high level of water. These findings demonstrate that organisms synthesizing chitin (but not HA) are capable of producing HA that is structurally and functionally relevant to that in mammals. The ability of insect cells to produce HA supports the idea that in vivo HA biosynthesis does not require molecules other than the HAS protein. An alternative model is that Drosophila cells use endogenous components of the chitin biosynthetic machinery to produce and secrete HA.

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.

The skeletal tissue development and patterning in chick limb buds are known to be under the spacio-temporal control of various heparin-binding cell growth factors such as fibroblast growth factors and bone morphogenetic proteins. Different structural regions on heparan sulfate (HS) chains of proteoglycans could be implicated in regional differences in the binding capacities of these cell growth factors, by which they could selectively interact with targeted cells and regulate their signaling in those processes. In this study we first demonstrated by cDNA cloning that one heparan sulfate 2-O-sulfotransferase (HS2ST) and two isoforms of heparan sulfate 6-O-sulfotransferase (HS6ST-1 and -2) occurred in chick embryos and had different substrate specificities each other. We next showed by whole mount in situ hybridization that the HS6ST-1 and HS6ST-2 transcripts were preferentially localized to the anterior proximal region and at the posterior proximal region of the limb bud, respectively, whereas the HS2ST transcript was distributed rather uniformly throughout the bud. Analyses of the structures of HS from different regions of the wing buds have shown variation in that 6-O-sulfated residues are more abundant in the proximal than distal region, whereas iduronosyl 6-O-sulfated residues are abundant in the anterior proximal region and glucuronosyl 6-O-sulfated residues in the posterior proximal region. These results suggest that HS with different sulfation patterns created with multiple sulfotransferase activities provides an appropriate extracellular environment for morphogenetic signal transduction.

Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank trade mark accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a beta 3-glycosyltransferase motif and a beta 4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUA beta 1-3GalNAc linkage and a GalNAc beta 1-4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1.

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.

We previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-alpha-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAP-to-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 x 106 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex.

Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing beta 3-glycosyltransferase motif but lacked a typical beta 4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUA beta 1-3GalNAc and GalNAc beta 1-4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant beta 4-glycosyltransferase architecture.

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912-37919). In the present study, we cloned putative catalytic domain of the human 3-OST-5 and expressed it in insect cells as a soluble enzyme. Recombinant 3-OST-5 only exhibited sulfotransferase activity toward heparan sulfate and heparin. When incubated heparan sulfate with [35S]PAPS, the highest incorporation of35S was observed, and digestion of the product with a mixture of heparin lyases yielded two major35S-labeled disaccharides, which were determined as DeltaHexA-GlcN(NS,3S,6S) and DeltaHexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of DeltaHexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcbeta1-3Gal1 structure (endo-beta-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 degrees C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function.

By a tblastn search with beta 1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in beta 1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5'-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger N-acetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly- and oligosaccharides, and chondroitin sulfate poly- and oligosaccharides with a beta 1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 transcripts were highly expressed in the small intestine, leukocytes, and spleen, however, both CSGalNAcTs were ubiquitously expressed in various tissues.

We found a novel human gene (GenBank accession number, Kazusa DNA Research Institute KIAA1402) that possesses homology with chondroitin synthase. The full-length open reading frame consists of 772 amino acids and encodes a typical type II membrane protein. This enzyme had a domain containing beta 3-glycosyltransferase motifs, which might be a beta3-glucuronyltransferase domain, but no domain with beta 4-glycosyltransferase motifs, although both are found in chondroitin synthase. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Its glucuronyltransferase activity was observed when chondroitin and chondroitin sulfate polysaccharides and oligosaccharides were used as acceptor substrates. However, it was not detected when dermatan sulfate, hyaluronan, heparan sulfate, heparin, N-acetylheparosan, lactosamine tetrasaccharide, and linkage tri- and tetrasaccharide acceptors were employed. The reaction product, which was speculated to exhibit a GlcA beta 1-3GalNAc linkage structure at its non-reducing terminus, showed the following characteristics. 1) It was catabolized by beta-glucuronidase. 2) It was an acceptor for Escherichia coli K4 chondroitin polymerase (K4 chondroitin polymerase). 3) The product of K4 chondroitin polymerase was cleaved by chondroitinase ACII. On the other hand, no N-acetylgalactosaminyltransferase activity was detected toward any acceptors. Quantitative real time PCR analysis revealed that its transcripts were highly expressed in the placenta, small intestine, and pancreas, although they were ubiquitously expressed in various tissues and cell lines. This enzyme could play a role in the synthesis of chondroitin sulfate as a glucuronyltransferase.

MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15-16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium.

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.

Serglycin is a family of small proteoglycans with Ser-Gly dipeptide repeats and is modified with various types of glycosaminoglycan side chains. We previously demonstrated that chondroitin sulfate-modified serglycin is a novel ligand for CD44 involved in the adherence and activation of lymphoid cells. In this study, we investigated the production and distribution of CD44 binding serglycins in various hematopoietic cells and characterized their carbohydrate side chains. Immunoprecipitation analysis using CD44-IgG and polyclonal antibody against the serglycin core peptide demonstrated that various serglycin species capable of binding CD44 are produced by a variety of hematopoietic cells including lymphoid cells, myeloid cells, and a few tumor cell lines. Glycosaminoglycans on these serglycins, which are essential for CD44 binding, are composed of chondroitin 4-sulfate or a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate, but no heparin or heparan sulfate side chain was detected. The serglycins are also secreted by normal splenocytes, lymph node lymphocytes, and bone marrow cells, whereas they are secreted in very small amounts by normal thymocytes. Secretion of serglycins is greatly enhanced by mitogenic stimulation with concanavalin A or lipopolysaccharide. Our results showed that serglycin, unlike hyaluronate, is produced and secreted in a functional (CD44 binding) form by many members of the hematopoietic system including various lymphocyte subsets. Our data suggest that serglycin may serve as a major ligand for CD44 in various events in the lymphohematopoietic system.

Heparan-sulfate 2-sulfotransferase (HS2ST), which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to L-iduronic acid at position 2 in heparan sulfate, was purified from cultured Chinese hamster ovary (CHO) cells to apparent homogeneity (Kobayashi, M., Habuchi, H., Habuchi, O., Saito, M., and Kimata, K. (1996) J. Biol. Chem. 271, 7645-7653). The internal amino acid sequences were obtained from the peptides after digestion of the purified protein with a combination of endoproteinases. Mixed oligonucleotides based on the peptide sequences were used as primers to obtain a probe fragment by reverse transcriptase-polymerase chain reaction using CHO cell poly(A)+ RNA as template. The clone obtained from a CHO cDNA library by screening with the probe is 2.2 kilobases in size and contains an open reading frame of 1068 bases encoding a new protein composed of 356 amino acid residues. The protein predicts a type II transmembrane topology similar to other Golgi membrane proteins. Messages of 5.0 and 3.0 kilobases were observed in Northern analysis. Evidence that the cDNA clone corresponds to the purified HS2ST protein is as follows. (a) The predicted amino acid sequence contains all five peptides obtained after endoproteinase digestion of the purified protein; (b) the characteristics of the predicted protein fit those of the purified protein in terms of molecular mass, membrane localization, and N-glycosylation; and (c) when the cDNA containing the entire coding sequence of the enzyme in a eukaryotic expression vector was transfected into COS-7 cells, the HS2ST activity increased 2.6-fold over controls, and the FLAG-HS2ST fusion protein purified by affinity chromatography showed the HS2ST activity alone.

We developed a mammalian transient expression system to isolate cDNA clones that determine hyaluronan expression. HAS-, a mouse mammary carcinoma mutant cell line, which is defective in hyaluronan synthase activity, was first established and used as a recipient for the expression cloning. One cloned cDNA that overcame the deficiency was isolated. The cDNA termed HAS contains an open reading frame of 1749 base pairs encoding a new protein of 583 amino acids. Homology analysis of the amino acid sequence suggests that HAS protein is related to streptococcal hyaluronan synthase and also to Xenopus laevis DG42 protein that was found to be homologous to bacterial hyaluronan synthase. Expression of HAS cDNA in HAS- cells complemented not only their mutant phenotypes such as deficient hyaluronan-matrix deposition but also hyaluronan synthase activity itself. Therefore, HAS cDNA is responsible for the activity of the hyaluronan synthase, a key enzyme of hyaluronan synthesis in eukaryotic cells.

Proteoglycans from rat liver had the ability to bind hepatocyte growth factor (HGF). Digestion of the proteoglycans with heparitinase resulted in the complete loss of the activity, while the digestion with chondroitinase ABC had no effect. Heparan sulfate (HS)-conjugated gel also bound HGF, and the binding was competitively inhibited by heparin and bovine liver HS, but not by Engelbreth-Holm-Swarm sarcoma HS, pig aorta HS, or other glycosaminoglycans, suggesting the specific structural domain in HS for the binding of HGF. Among limited digests with heparitinase I of bovine liver HS, octasaccharide is the minimal size to bind HGF. Comparison of the disaccharide unit compositions revealed a marked difference in IdoA(2SO4)-GlcNSO3(6SO4) unit between the bound and unbound octasaccharides. The contents of this disaccharide unit were calculated to be 2 mol/mol for the bound octasaccharide but 1 mol/mol for the unbound one. Considering both the substrate specificity and properties of heparitinase I, the above results suggest that the bound octasaccharide should contain two units of IdoA(2SO4)-GlcNSO3(6SO4) contiguously or alternately in the vicinity of the reducing end. The bound decasaccharide was more than 20 times as active as the unbound one with regard to the ability to release HGF bound to rat liver HS proteoglycan. The ability was comparable to the one-fourth of that of heparin.

We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-alpha-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP.HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP.HA complex was digested with thermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP.HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-alpha-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-alpha-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the C-terminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.44 microM.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

PG-M is a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds. We previously isolated cDNA clones encoding the core protein of PG-M (Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469). The amino acid sequence deduced from the cDNA analysis revealed the presence of two epidermal growth factor-like domains, a C-type lectin-like domain, and a complement regulatory protein (CRP)-like domain at the COOH terminus. The COOH-terminal portion has been expressed as a fusion protein with glutathione S-transferase in Escherichia coli to test its carbohydrate binding activity using affinity chromatography. The purified fusion protein binds to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner. Furthermore, the fusion protein binds to heparin- or heparan sulfate-Sepharose. To investigate roles of each COOH-terminal domain, we have made a truncated construct which lacks the CRP-like domain and determined if the CRP-like domain is involved in the binding activity. The removal of this domain resulted in the complete loss of both C-type lectin-like and heparin binding activities. The results suggest that a whole set of epidermal growth factor-, lectin-, and CRP-like domains may serve a functional structure for these bindings.

We showed previously that hyaluronan (HA) synthesized by cultured fibroblasts firmly bound 85-kDa proteins. The proteins were derived from serum used for the culture and appeared to be covalently linked to HA (Yoneda, M., Suzuki, S., and Kimata, K. (1990) J. Biol. Chem. 265, 5247-5257). In these regards, we named this molecule SHAP (serum-derived HA associated proteins). Incubation of serum with exogenous HA under physiological conditions enabled us to prepare SHAP.HA complex without cell cultivation. The complex thus obtained from bovine or human serum was served for the characterization of SHAP. Digestion with HA-lyase and subsequent separation on SDS-polyacrylamide gel electrophoresis yielded two components, X and Y. Because of the block of their NH2 termini, peptides were obtained by the digestion of X and Y with V8 protease, separated on SDS-polyacryl-amide gel electrophoresis and then subjected to the analysis. Peptides from X and Y showed a high degree of sequence similarity to the two heavy chains, HC2 and HC1, of human inter-alpha-trypsin inhibitor (ITI), respectively (over 80% with bovine SHAP and essentially 100% with human SHAP). Cross-reactivity with antibodies against ITI supported the findings. Direct digestion of the complex with V8 protease and the subsequent purification of the HA-resistant fragment complex were performed to identify the HA-binding domains. NH2-terminal sequences of the fragments suggested the participation of the COOH-terminal half of ITI with an amphipathic alpha helix structure in the HA binding.

We have isolated cDNA clones encoding the core protein of PG-M, a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds (Shinomura T., Jensen, K. L., Yamagata, M., Kimata, K., and Solursh, M. (1990) Anat. Embryol. 181, 227-233). The amino acid sequence deduced from the cDNA analysis revealed the presence of a hyaluronic acid binding domain at the amino-terminal side and two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like domain at the carboxyl-terminal side. These domains show an extremely high homology to corresponding domains of a human fibroblast large chondroitin sulfate proteoglycan, versican. Such evolutionally conserved structures in the PG-M core protein might be involved in important biological functions of this molecule. On the other hand, the chondroitin sulfate attachment domain at the middle region of the PG-M core protein shows no significant amino acid sequence homology to the corresponding domain of the versican core protein. Further, the chondroitin sulfate attachment domain of PG-M core protein is about 100 kDa larger than that of versican core protein. The finding of alternatively spliced forms of the PG-M core protein suggests that versican might be one of the multiple forms of PG-M.

We have isolated cDNA clones encoding the core protein of PG-Lb, proteoglycan which has been shown to be preferentially expressed in the zone of flattened chondrocytes of the developing chick limb cartilage (Shinomura, T., Kimata, K., Oike, Y., Yano, S., and Suzuki, S. (1984) Dev. Biol. 103, 211-220). The deduced amino acid sequence from the cDNA analysis indicates the presence of consensus leucine-rich repeats which are present in other small proteoglycans, decorin, biglycan, and fibromodulin. However, the homology analysis revealed that chick PG-Lb showed a higher homology (about 50% in the region containing leucine-rich repeats) to human osteoinductive factor, OIF, rather than to the other small proteoglycans. Furthermore, 6 cysteine residues are detected in both PG-Lb and OIF with invariant relative positions. Therefore, such an evolutionarily conserved structure in the PG-Lb core protein might be involved in some important biological functions of this molecule. In close relation to the structural similarity to OIF, the unique expression of PG-Lb in the ossifying area of cartilage suggested the possible participation of this proteoglycan in osteogenic processes.

1. Formation of 11 sugar nucleotides from the respective nucleoside triphosphates and sugar 1-phosphates have been demonstrated with the crude extract of Azotobacter vinelandii. These are adenosine diphosphate glucose, cytidine diphosphate glucose, uridine diphosphate glucose, deoxythymidine diphosphate glucose, adenosine diphosphate N-acetylglucosamine, uridine diphosphate N-acetylglucosamine, deoxythymidine diphosphate N-acetylglucosamine, adenosine diphosphate glucosamine, cytidine diphosphate glucosamine, uridine diphosphate glucosamine, and deoxythymidine diphosphate glucosamine. 2. The CDP-glucose pyrophosphorylase activity has been resolved into two peaks (Fractions I and II) by diethylaminoethyl cellulose chromatography. 3. One of these fractions (Fraction I: Michaelis constant (Km) for cytidine triphosphate = 3.3 x 10-3 m) is accompanied by a predominant UDP-glucosamine pyrophosphorylase activity (Km for uridine triphosphate = 1.9 x 10-4 m), and the ratio of the two activities (1:26) did not vary during the purification and inactivation at 50°. It seems likely that both activities are catalyzed by the same enzyme. 4. Another fraction (Fraction II) has been purified 195-fold. The purified enzyme is specific for both nucleoside triphosphate and sugar 1-phosphate except glucosamine 1-phosphate, which is active in replacing the glucose 1-phosphate. The pH optimum of the reaction is 8.5, and its equilibrium constant measured in the direction of CDP-glucose synthesis is 0.57. The enzyme absolutely requires Mg++ ions for activity. Variation of the Mg++ ion concentration relative to either pyrophosphate or CTP has a marked influence on the reaction velocity as well as Km values for the substrates. Km values calculated for pyrophosphate and CTP are 5.6 x 10-4 m and 7 x 10-4 m, respectively, when the molar ratio of MgCl2 to the substrate is 2:1. 5. The enzyme (Fraction II) is inhibited by its products, CDP-glucose and pyrophosphate. The inhibition by CDP-glucose is strictly competitive with respect to CTP, whereas that by pyrophosphate is noncompetitive. 6. Deoxythymidine triphosphate is a strong inhibitor of the enzyme (Fraction II). It is competitive with CTP only partially, indicating that dTTP combines with a site other than the CTP-binding site. 7. The cytidine nucleotides, isolated from A. vinelandii and tentatively identified as cytidine diphosphate 2-O-methyldeoxyaldose and its carboxylic acid ester, also inhibit the enzyme (Fraction II). Plots of percentage inhibition against inhibitor concentration give sigmoid curves.