Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.

Two novel chondroitinases, chondroitin ABC lyase (EC 4.2.2.4) and chondroitin AC lyase (EC 4.2.2.5), have been purified from Bacteroides stercoris HJ-15, which was isolated from human intestinal bacteria with glycosaminoglycan degrading enzymes. Chondroitin ABC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and Sephacryl S-300 column chromatography with a final specific activity of 45.7 micromol.min-1.mg-1. Chondroitin AC lyase was purified to apparent homogeneity by a combination of QAE-cellulose, CM-Sephadex C-50, hydroxyapatite and phosphocellulose column chromatography with a final specific activity of 57.03 micromol.min-1.mg-1. Chondroitin ABC lyase is a single subunit of 116 kDa by SDS/PAGE and gel filtration. Chondroitin AC lyase is composed of two identical subunits of 84 kDa by SDS/PAGE and gel filtration. Chondroitin ABC and AC lyases showed optimal activity at pH 7.0 and 40 degrees C, and 5.7-6.0 and 45-50 degrees C, respectively. Both chondroitin lyases were potently inhibited by Cu2+, Zn2+, and p-chloromercuriphenyl sulfonic acid. The purified Bacteroidal chondroitin ABC lyase acted to the greatest extent on chondroitin sulfate A (chondroitin 4-sulfate), to a lesser extent on chondroitin sulfate B (dermatan sulfate) and C (chondroitin 6-sulfate). The purified chondroitin AC lyase acted to the greatest extent on chondroitin sulfate A, and to a lesser extent on chondroitin C and hyaluronic acid. They did not act on heparin and heparan sulfate. These findings suggest that the biochemical properties of these purified chondroitin lyases are different from those of the previously purified chondroitin lyases.     

Principles of mimicking and engineering the self-organized structure of hard tissues

The mechanism of the formation of a self-aligned hydroxyapatite (HAP) nanocrystallite structure was examined. It is found that the highly ordered HAP nanocrystallite assembly is attributed to the so-called self-(homo)epitaxial nucleation and growth. On the other hand, according to this mechanism, a high supersaturation will give rise to a random assembly of HAP crystallites. The effects of ions, biosubstrate, and supersaturation on the micro/nanostructure correlation between substrate and biominerals as well as their implications in hard tissue formation were examined. Surprisingly, some biomolecules are found to be able to suppress the supersaturation-driven interfacial structure mismatch and hence promote the well aligned HAP pattern formation.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.     

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate

In the preceding paper (Inoue, H., Otsu, K., Yoneda, M., Kimata, K., Suzuki, S., and Nakanishi, Y. (1986) J. Biol. Chem. 261, 4460-4469), we reported the purification from human serum of an N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase fraction which was able to transfer sulfate predominantly to position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin sulfate. We now show that the activity toward the terminal was co-purified with a minor activity toward the interior counterpart by sequential chromatography on heparin-Sepharose CL-6B, Matrex Blue B, hydroxyapatite, and Sephacryl S-300, and that the two activities were equally heatlabile. The enzyme purified 5000-fold from human serum was devoid of the sulfotransferase activities toward chondroitin, heparan sulfate, and keratan sulfate, but showed a strong terminal sulfotransferase activity toward dermatan sulfate (pig skin); over 97% of the sulfate residues incorporated were at position 6 of the nonreducing N-acetylgalactosamine 4,6-bissulfate end groups linked to the L-iduronic acid group. Although the enzyme introduces sulfate predominantly into the nonreducing terminal of chondroitin sulfate at physiological pH (approximately equal to 7.0) and Ca2+ concentration (approximately 2-3 mM), the activity toward the interior portion relative to that toward the terminal was increased by either lowering pH or elevating Ca2+ concentration, perhaps owing to changes in the conformation or ionic state of the acceptor molecule. Comparison between the human serum enzyme and the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (formerly designated "E6-sulfotransferase") from squid cartilage indicated that the latter is distinct from the former in introducing sulfate predominantly into the interior portion of chondroitin sulfate. It appears that the role of the squid sulfotransferase is to synthesize so-called chondroitin sulfate E where over 50% of the interior hexosamine units are 4,6-bis-sulfated.     

Liver alcohol dehydrogenase subunit equivalence studied by rapid sampling of alcohol product formed from sequentially bound [4alpha-3H]NADH.

Proteoglycans of calf and steer articular cartilage were studied with a view of assessing structure and changes occurring as a result of the aging process. The average reduction in hydrodynamic size noted in steer was associated with a diminution in size of the chondroitin sulfate-rich region of the core protein as well as the chondroitin sulfate chains themselves. By contrast the keratan sulfate-rich region was hydrodynamically larger in steer although the keratan sulfate chains were only slightly longer than in calf. The proteoglycans showed a maturation-related decrease in chondroitin sulfate content (shorter chains, fewer chains, smaller chondroitin sulfate-rich region) and an enrichment in keratan sulfate chains in both the chondroitin sulfate-rich and keratan sulfate-rich regions. Proteoglycans from both age groups contained an oligosaccharide which was recovered mainly from outside of the keratan sulfate-rich region. There were no significant differences in size between keratan sulfate chains recovered from the keratan sulfate-rich region and the chondroitin sulfate-rich region.     

Elimination of breast tumor-associated chondroitin sulfate promotes metastasis

Breast cancer is one of the leading causes of cancer-related deaths amongst women in the USA. The tumor microenvironment has been suggested to be an attractive therapeutic target for treatment of cancers. The glycosaminoglycan chondroitin sulfate, as part of the cellular microenvironment, consists of long linear chains of repeating disaccharide units, which are covalently attached to core proteins to form chondroitin sulfate-proteoglycans. In vitro studies have implicated chondroitin sulfate in various aspects of carcinogenesis, whereas the in vivo roles of chondroitin sulfate are less clear. Drastically elevated levels of chondroitin sulfate have been observed within the stromal compartment of many solid tumors, including human breast carcinomas, the significance of which is unknown. We examined the role of tumor-associated chondroitin sulfate in breast cancer progression. Enzymatic elimination of endogenous chondroitin sulfate by intra-tumor injections of chondroitinase ABC leads to the development of secondary tumors and increased lung metastases, while primary orthotopic tumor growth was not affected. These results establish a metastasis-inhibiting effect of primary breast tumor-associated chondroitin sulfate, which may open novel carbohydrate-based therapeutic strategies to combat breast cancer.     

Ultrastructural and immunocytochemical study of bone-derived cells cultured in three-dimensional matrices: Influence of chondroitin-4 sulfate on mineralization

Abstract. Bone-derived cells were cultured in three-dimensional reconstituted matrices made of type I collagen or type I collagen chondroitin-4-sulfate. As observed by microscope, their characteristics were as follows: The cells deposited a faint extracellular matrix mainly composed of type I collagen. In the collagen-chondroitin-sulfate sponge fibers, a calcification process, which involved the deposition of hydroxyapatite crystals, was demonstrated. Mineralization occurred only in collagen chondroitin sulfate sponge fibers when seeded with bone-derived cells and was not seen with nonosteogenic cells, such as gingival fibroblasts. Gla protein was intracellularly visualized in both types of sponges seeded with bone-derived cells while an extracellular secretion was seen only in the collagen chondroitin sulfate sponge fibers where calcification occurred. These results suggest that collagen chondroitin sulfate promotes in vitro mineralization of three-dimensional collagen matrices when seeded with bone-derived cells.     

Posttranslational modifications to human bone sialoprotein determined by mass spectrometry.

Bone sialoprotein (BSP) is an acidic 301 amino acid protein expressed by osteoblasts and at a low level by hypertrophic chondrocytes. Its expression is highest during early stages of bone formation, and it is particularly abundant in the cells lining the surface of newly formed trabeculae. BSP contains numerous substituents which are anionic in nature and apparently essential for the function of the protein. Thus, the proposed role of BSP in hydroxyapatite nucleation and growth may depend on such modifying groups. The posttranslational modifications include several acidic oligosaccharides as well as phosphate and sulfate groups. This work combines matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with selective enzyme treatment of BSP to provide new information on the precise distribution and structure of oligosaccharides, sulfate, and phosphate groups in BSP isolated from human bone. The results provide a high level of detail in the location of these modifying groups toward the end of providing a basis for further understanding the function of BSP in bone nucleation.     

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-β1 expression in mesangial cells: biochemical and structural considerations

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-β1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-β1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-β1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-β1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-β1 overexpression.     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.     

Synthesis and characterizations of amphiphilic poly(L-lactide)-grafted chondroitin sulfate copolymer and its application as drug carrier

In this investigation, new biodegradable brush-like amphiphilic copolymers were synthesized by ring opening polymerization. Poly(L-lactide) (PLLA) was grafted onto chondroitin sulfate (CS), which is one of the physiologically significant specific glycosaminoglycans (GAGs), using a tin octanoate [Sn(Oct)2] catalyst in DMSO. The hydroxyl groups of the chondroitin sulfate were used as initiating groups. These functional groups enable specific mucoadhesion or receptor recognition. The degree of substitution (DS), the degree of polymerization (DP) and the chondroitin sulfate content (from 1.1 to 15.4%) were analyzed by 1H NMR. The characteristics of these grafted copolymers, including the structure, the thermal properties and biodegradability, etc., were examined with respect to CS content. Meanwhile, the amphiphilic core (PLLA)-corona (CS) nanoparticles, with size smaller than 200 nm, was examined by dynamic light scattering (DLS). Zeta potential analysis exhibited the value in the range -18.3 to -49.4 mV. The morphologies of the nanoparticles were observed by field-emission scanning electron microscopy (FE-SEM). The nanoparticles with lower cytotoxicity were examined by MTT assay. Furthermore, the in vitro BSA release kinetics of those CSn-PLLA nanoparticles was also determined in this study.     

Kinetic analysis of protein crystal nucleation in gel matrix

The effect of agarose on nucleation of hen egg white lysozyme crystal was examined quantitatively using a temperature-jumping technique. For the first time, to our knowledge, the inhibition of agarose during the nucleation of lysozyme was quantified in two respects: a), the effect of increasing interfacial nucleation barrier, described by the so-called interfacial correlation parameter f(m); and b), the ratio of diffusion to interfacial kinetics obtained from dynamic surface tension measurements. It follows from a dynamic surface tension analysis that the agarose network inhibits the nucleation of lysozyme by means of an enhancement of the repulsion and interfacial structure mismatch between foreign bodies and lysozyme crystals, slowing down the diffusion process of the protein molecules and clusters toward the crystal-fluid interface and inhibiting the rearrangement of protein molecules at the interface. Our results, based on ultraviolet-visible spectroscopy, also show no evidence of the supersaturation enhancement effect in protein agarose gels. The effects of nucleation suppression and transport limitation in gels result in bigger, fewer, and perhaps better quality protein crystals. The understandings obtained in this study will improve our knowledge in controlling the crystallization of proteins and other biomolecules.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

Altered cartilage proteoglycans synthesized by chick limb bud chondrocytes cultured in serum-free defined medium

Chick high-density culture chondrocytes synthesize cartilage-specific proteoglycans with much structural similarity to the proteoglycans made by cartilage in vivo. Such cultures can be maintained in a defined medium formulated in this laboratory in which chondrogenesis occurs without the addition of serum. The proteoglycans synthesized by the chondrocytes in the presence of defined medium are of a cartilage-specific structure but differ in some aspects from the proteoglycans made in serum-containing medium. While their buoyant density, ability to aggregate with hyaluronic acid, and keratan sulfate chain size are unchanged, the proteoglycans synthesized in defined medium have altered chondroitin sulfate chains. This chondroitin sulfate is of significantly larger size and has a different sulfation pattern relative to that produced in serum-containing medium. The larger size of the chondroitin sulfate results in a larger monomer size of the defined medium proteoglycans. These differences have implications about the regulation of the structure of chondroitin sulfate proteoglycans.     

Utilization of Chondroitin Sulfate by Bacteroides thetaiotaomicron Growing in Carbohydrate-Limited Continuous Culture

When Bacteroides thetaiotaomicron, an obligate anaerobe from the human colonic flora, was grown in continuous culture with the mucopolysaccharide chondroitin sulfate as the limiting source of carbohydrate, growth yields ranged from 48 g of cell dry weight per mol of equivalent monosaccharide at a growth rate of 3.5 h per generation to 32 g per mol at a growth rate of 24 h per generation. The theoretical maximum growth yield (61 g of cell dry weight per mol of equivalent monosaccharide) was comparable to that of 54 g per mol, which was obtained previously when glucuronic acid, a component of chondroitin sulfate, was the limiting carbohydrate (S. F. Kotarski and A. A. Salyers, J. Bacteriol. 146:853-860, 1981). However, the maintenance coefficient was three times higher when chondroitin sulfate was the substrate than when glucuronic acid was the substrate. The specific activity of chondroitin lyase (EC 4.2.2.4), an enzyme which cleaves chondroitin sulfate into disaccharides, declined by nearly 50% as growth rates decreased from 3.5 to 24 h per generation. By contrast, the specific activities of several glycolytic enzymes and disaccharidases remained constant over this range of growth rates. Although chondroitin sulfate was growth limiting, some carbohydrate was detectable in the extracellular fluid at all growth rates. At rapid growth rates (1 to 2 h per generation), this residual carbohydrate included fragments of chondroitin sulfate having a wide range of molecular weights. At slower growth rates (2 to 24 h per generation), the residual carbohydrate consisted mainly of a small fragment which migrated on paper chromatograms more slowly than the disaccharides produced by chondroitin lyase but faster than a tetrasaccharide. This small fragment may represent the reducing end of the chondroitin sulfate molecule.     

Fractionation and isolation of heparan sulfates using poly-L-lysine-Sepharose

A simple procedure for the isolation of heparan sulfates from pig lung using a poly-L-lysine-Sepharose column is described. Glycosaminoglycans are absorbed on poly-L-lysine-Sepharose at pH 7.5 and eluted with an NaCl linear gradient in the following order: hyaluronic acid (0.32 M NaCl), chondroitin (0.36 M NaCl), keratan sulfate (0.80 M NaCl), chondroitin 4-sulfate (0.86 M NaCl), chondroitin 6-sulfate (0.95 M NaCl), dermatan sulfate (0.91 M NaCl), heparan sulfate (1.2 M NaCl), and heparin (1.35 M NaCl). Based on these observations, isolation of heparan sulfate from pig lung crude heparan sulfate fractions which contain chondroitin sulfates and dermatan sulfate was attempted, using this chromatographic technique.     

硫酸软骨素在癌症治疗领域的研究进展

硫酸软骨素是一种硫酸化的糖胺聚糖,其在恶性肿瘤组织中的含量、结构、硫酸化位点等与正常组织存在显著差异,在癌症的迁移,侵袭,血管生成过程中发挥重要调控作用,在癌症的临床研究中具有很大潜力。该文对硫酸软骨素的生物合成进行归类分析,对近几年硫酸软骨素与肿瘤入侵和转移的相关临床研究以及分子机制研究做出综述,以期为开发硫酸软骨素潜在的临床价值和肿瘤治疗靶点研究提供理论依据,为恶性肿瘤的早期诊断和预后评估提供思路。     

Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate.

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.