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Osami Habuchi

Publications and source records attributed to Osami Habuchi.

14 recordsLinked to original sources
PubMed2006

N-linked oligosaccharides on chondroitin 6-sulfotransferase-1 are required for production of the active enzyme, Golgi localization, and sulfotransferase activity toward keratan sulfate.

We have shown previously that purified chondroitin 6-sulfotransferase-1 (C6ST-1) was a glycoprotein abundant in N-linked oligosaccharides and could sulfate both chondroitin (C6ST activity) and keratan sulfate (KSST activity); however, functional roles of the N-glycans have remained unclear. In the present study, we show essential roles of N-glycans attached to C6ST-1 in the generation of the active enzyme and in its KSST activity. Treatment with tunicamycin of COS-7 cells transfected with C6ST-1 cDNA totally abolished production of the active C6ST-1. A nearly complete removal of N-glycans of the recombinant C6ST-1 by peptide N-glycosidase F increased the C6ST activity but decreased the KSST activity. Among six potential N-glycosylation sites, deletion of the fourth or sixth site from the amino terminus inhibited production of the active C6ST-1, whereas deletion of the fifth site resulted in a marked loss of the KSST activity. Wild-type recombinant C6ST-1 showed a typical Golgi localization, whereas M-4 recombinant C6ST-1, in which the fourth N-glycosylation site was deleted, colocalized with calnexin, an endoplasmic reticulum-resident protein. Unlike wildtype recombinant C6ST-1, M-4 recombinant C6ST-1 showed a weak affinity toward wheat germ agglutinin and was converted completely to the nonglycosylated form by endoglycosidase H. These observations suggest that N-glycan attached to the fourth N-glycosylation site may function in the proper processing of N-glycans required for the Golgi localization, thereby causing the production of the active C6ST-1, and that N-glycan attached to the fifth N-glycosylation site may contribute to the KSST activity of C6ST-1.

Amino Acid Sequence↗
PubMed2006

Determination of substrate specificity of sulfotransferases and glycosyltransferases (proteoglycans).

Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high-performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2-sulfotransferase, heparan sulfate 6-sulfotransferases, chondroitin 4-sulfotransferases, chondroitin 6-sulfotransferase, N-acetylgalactosamine 4-sulfate 6-sulfotransferase, and N-acetylglucosamine 6-sulfotransferases.

Deamination↗
PubMed2005

Recognition of sulfation pattern of chondroitin sulfate by uronosyl 2-O-sulfotransferase.

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.

Animals↗
PubMed2005

Synthesis of sulfated phenyl 2-acetamido-2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl 2-acetamido-2-deoxy-beta-D-galactopyranoside is a competitive acceptor that decreases sulfation of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase.

We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO(4)) repeating units. To investigate the function of chondroitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl alpha- or -beta-2-acetamido-2-deoxy-beta-D-galactopyranosides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant GalNAc4S-6ST. We found that phenyl beta-GalNAc(4SO(4)) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The sulfated product derived from phenyl beta-GalNAc(4SO(4)) was identical to phenyl beta-GalNAc(4,6-SO(4)). These observations indicate that derivatives of beta-D-GalNAc(4SO(4)) are possible specific inhibitors of GalNAc4S-6ST.

Acetylgalactosamine↗
PubMed2005

N-linked oligosaccharides are required to produce and stabilize the active form of chondroitin 4-sulphotransferase-1.

C4ST-1 (chondroitin 4-sulphotransferase-1) transfers sulphate to position 4 of N-acetylgalactosamine in chondroitin. We showed previously that purified C4ST-1 from the culture medium of rat chondrosarcoma cells was a glycoprotein containing approx. 35% N-linked oligosaccharides. In the present paper, we investigated the functional role of the N-linked oligosaccharides attached to C4ST-1. We found that (i) treatment of recombinant C4ST-1 with peptide N-glycosidase F caused a marked decrease in activity, (ii) production of the active form of C4ST-1 by COS-7 cells transfected with cDNA of C4ST-1 was inhibited by tunicamycin, (iii) deletion of the N-glycosylation site located at the C-terminal region of C4ST-1 abolished activity, (iv) attachment of a single N-glycan at the C-terminal region supported production of the active form of C4ST-1, but the resulting recombinant enzyme was much more unstable at 37 degrees C than the control recombinant protein, and (v) truncation of C-terminal region up to the N-glycosylation site at the C-terminal region resulted in total loss of activity. These observations strongly suggest that N-linked oligosaccharides attached to C4ST-1 contribute to the production and stability of the active form of C4ST-1. In addition, the N-linked oligosaccharide at the C-terminal region appears to affect the glycosylation pattern of recombinant C4ST; a broad protein band of the wildtype protein resulting from microheterogeneity of N-linked oligosaccharides disappeared and four discrete protein bands with different numbers of N-linked oligosaccharides appeared when the N-linked oligosaccharide at the C-terminal region was deleted.

Animals↗
PubMed2004

Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighbouring the acceptor GalNAc residues.

C4ST-1 (chondroitin 4-sulphotransferase-1) and C6ST-1 (chondroitin 6-sulphotransferase-1) transfer sulphate from PAPS (adenosine 3'-phosphate 5'-phosphosulphate) to positions 4 and 6 respectively of the GalNAc residues of chondroitin. We showed previously that C4ST-1 purified from rat chondrosarcoma and recombinant C4ST-1 both transfer sulphate efficiently to position 4 of the GalNAc residues of DSDS (desulphated dermatan sulphate). We report here the specificity of C4ST-1 and C6ST-1 in terms of uronic acid residue recognition around the GalNAc residue to which sulphate is transferred. When [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C4ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in disaccharide fractions and the remainder distributed to tetrasaccharides and larger fractions, indicating that C4ST-1 mainly transferred sulphate to position 4 of the GalNAc residue located at the GlcA-GalNAc-GlcA sequence. Structural analysis of tetrasaccharide and larger oligosaccharide fractions indicated that C4ST-1 mainly transferred sulphate to the GalNAc residue adjacent to the reducing side of the GlcA residue. On the other hand, when [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C6ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in fractions larger than hexasaccharides, indicating that C6ST-1 transferred sulphate to the GalNAc residues located in the L-iduronic acid-rich region. Structural analysis of the tetrasaccharide and larger oligosaccharide fractions indicated that C6ST-1 showed very little preference for the GalNAc residue neighbouring the GlcA residue. These results indicate that C4ST-1 and C6ST-1 differ from each other in the recognition of uronic acid residues adjacent to the targeted GalNAc residue.

Acetylgalactosamine↗
PubMed2004

Sulfation pattern in glycosaminoglycan: does it have a code?

Heparan sulfate chains (HS) are initially synthesized on core proteins as linear polysaccharides composed of glucuronic acid--N-acetylglucosamine repeating units and subjected to marked structural modification by sulfation (N-, 2-O-, 6-O-, 3-O-sulfotransferases) and epimerization (C5-epimerase) at the Golgi lumen and further by desulfation (6-O- endosulfatase) at the cell surface, after which divergent fine structures are generated. The expression patterns and specificity of the modifying enzymes are, at least partly, responsible for the elaboration of these fine structures of heparan sulfate. HS interacts with many proteins including growth factors (GF) and morphogens through specific fine structures. Recent biochemical and genetic studies have presented evidence that HS plays important roles in cell behavior and organogenesis. In knock-down experiments of heparan sulfate 6-O-sulfotransferase, 6-O-sulfated units in HS have been shown to act as a stimulator or suppressor according to individual GF/morphogen signaling systems.

Animals↗
PubMed2003

A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase.

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.

Animals↗
PubMed2003

Biosynthesis of heparan sulphate with diverse structures and functions: two alternatively spliced forms of human heparan sulphate 6-O-sulphotransferase-2 having different expression patterns and properties.

Heparan sulphate 6- O -sulphotransferase (HS6ST) catalyses the transfer of sulphate from adenosine 3'-phosphate, 5'-phosphosulphate to the 6th position of the N -sulphoglucosamine residue in HS. We previously described the occurrence of three isoforms of mouse HS6ST, mHS6ST-1, -2, and -3 [Habuchi, Tanaka, Habuchi, Yoshida, Suzuki, Ban and Kimata (2000) J. Biol. Chem. 275, 2859-2868]. In the present study, we have characterized HS6ST-2 and HS6ST-1 human isologues, including their chromosomal localizations. In the process of their cDNA cloning, we found two forms of HS6ST-2: the original (hHS6ST-2) and a short form (hHS6ST-2S) with 40 amino acids deleted. Both hHS6ST-2 and hHS6ST-2S catalysed the same sulphation reaction, but their preferences for sulphation sites in HS substrates were different. Dot-blot analysis of the two forms showed that the original form was exclusively expressed in adult and foetal brain tissues, whereas the short form was expressed preferentially in ovary, placenta and foetal kidney, suggesting that the expression of two forms of hHS6ST-2 is strictly regulated to yield tissue-dependent differences in the fine structure of HS. A refined analysis of their reaction products has led us to another finding, that HS6STs could also transfer sulphate to N -sulphoglucosamine residues located at the non-reducing terminal of HS with high affinity.

Alternative Splicing↗
PubMed2003

Mouse N-acetylgalactosamine 4-sulfotransferases-1 and -2. Molecular cloning, expression, chromosomal mapping and detection of their activity with GalNAcbeta1-4GlcNAcbeta1-octyl.

N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcbeta1-4GlcNAcbeta1-octyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal-B5 proximal and chromosome 18A2 distal-B1 proximal, respectively.

Acetylgalactosamine↗
PubMed2002

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage.

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.

Animals↗
PubMed2002

Glycosaminoglycan structures required for strong binding to midkine, a heparin-binding growth factor.

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.

Animals↗
PubMed2001

Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st(-/-)) by embryonic stem cell technology. C6st(-/-) mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st(-/-) mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st(-/-) mice. Further analysis revealed that the number of CD62L(+)CD44(low) T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st(-/-) mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.

Animals↗