GlcA Residues Research Articles

LARGE2 generates the same xylose- and glucuronic acid-containing glycan structures as LARGE

LARGE (like-glycosyltransferase) and LARGE2 (glycosyltransferase-like 1B (GYLTL1B)) are homologous Golgi glycosyltransferases possessing two catalytic domains with homology to members of glycosyltransferase families GT8 and GT49. Mutations in human and mouse Large result in muscular dystrophy due to underglycosylation of dystroglycan. The systemic function of LARGE2 is unknown, but at a cellular level the enzyme can substitute for LARGE in glycosylating dystroglycan. Here, we show that LARGE2 catalyzes the same glycosylation reaction as LARGE. It is a bifunctional glycosyltransferase using uridine diphosphate (UDP)-xylose (Xyl) and UDP-glucuronic acid (GlcA) as donor sugars to produce a xyloglucuronan with alternating Xyl and GlcA residues.

Three Arabidopsis DUF579 Domain-Containing GXM Proteins are Methyltransferases Catalyzing 4-O-Methylation of Glucuronic Acid on Xylan

Xylan is made of a linear chain of β-1,4-linked xylosyl residues, some of which are substituted with side chains, such as glucuronic acid (GlcA), methylglucuronic acid (MeGlcA) and arabinose, depending on the source of xylan. Although past studies have revealed a number of genes involved in the elongation of the xylan backbone and the addition of GlcA and arabinosyl side chains, no genes have been shown to be implicated in glucuronoxylan methylation. In this report, we investigated the roles of three Arabidopsis genes, namely GLUCURONOXYLAN METHYLTRANSFERASE1 (GXM1), GXM2 and GXM3, in xylan biosynthesis. The GXM1/2/3 genes were found to be expressed in secondary wall-forming cells and their expression was regulated by SND1, a secondary wall master transcriptional switch. Their encoded proteins were shown to be located in the Golgi, where xylan biosynthesis occurs. Chemical analysis of cell wall sugars from single and double mutants of these genes revealed that although no alterations in the amount of xylose were observed, a significant reduction in the level of MeGlcA was evident in the gxm3 single mutant and the gxm double mutants. Structural analysis of xylan demonstrated that the gxm mutations caused a specific defect in GlcA methylation on xylan without affecting the frequency of xylan substitution. Only about 10% of the GlcA residues on xylan were methylated in the gxm2/3 double mutant, whereas in the wild type 60% of the GlcA residues were methylated. Furthermore, an activity assay demonstrated that recombinant GXM proteins exhibited a methyltransferase activity capable of transferring the methyl group from S-adenosylmethionine onto GlcA-substituted xylooligomers and simultaneous mutations of GXM2/3 genes caused a loss of such a methyltransferase activity. Taken together, our results provide the first line of genetic and biochemical evidence that the three DUF579 domain-containing proteins, GXM1, GXM2 and GXM3, are methyltransferases catalyzing 4-O-methylation of GlcA side chains on xylan.

Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis

Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.

Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that mutations of two Arabidopsis family GT8 genes, GUX1 and GUX2, affect the addition of GlcA and MeGlcA to xylan, but it is not known whether they encode glucuronyltransferases (GlcATs) or indirectly regulate the GlcAT activity. In this study, we performed biochemical and genetic analyses of three Arabidopsis GUX genes to determine their roles in the GlcA substitution of xylan and secondary wall deposition. The GUX1/2/3 genes were found to be expressed in interfascicular fibers and xylem cells, the two major types of secondary wall-containing cells that have abundant xylan. When expressed in tobacco BY2 cells, the GUX1/2/3 proteins exhibited an activity capable of transferring GlcA residues from the UDP-GlcA donor onto xylooligomer acceptors, demonstrating that these GUX proteins possess xylan GlcAT activity. Analyses of the single, double and triple gux mutants revealed that simultaneous mutations of all three GUX genes led to a complete loss of GlcA and MeGlcA side chains on xylan, indicating that all three GUX proteins are involved in the GlcA substitution of xylan. Furthermore, a complete loss of GlcA and MeGlcA side chains in the gux1/2/3 triple mutant resulted in reduced secondary wall thickening, collapsed vessel morphology and reduced plant growth. Together, our results provide biochemical and genetic evidence that GUX1/2/3 are GlcATs responsible for the GlcA substitution of xylan, which is essential for normal secondary wall deposition and plant development.

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour ( Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)- d-galactan backbone with β-(1→6)- d-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α- l-Ara f. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β- l-Ara p. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

K5 lyase A (KflA) is a tail spike protein (TSP) encoded by a K5A coliphage, which cleaves K5 capsular polysaccharide, a glycosaminoglycan with the repeat unit [-4)-betaGlcA-(1,4)- alphaGlcNAc(1-], displayed on the surface of Escherichia coli K5 strains. The crystal structure of KflA reveals a trimeric arrangement, with each monomer containing a right-handed, single-stranded parallel beta-helix domain. Stable trimer formation by the intertwining of strands in the C-terminal domain, followed by proteolytic maturation, is likely to be catalyzed by an autochaperone as described for K1F endosialidase. The structure of KflA represents the first bacteriophage tail spike protein combining polysaccharide lyase activity with a single-stranded parallel beta-helix fold. We propose a catalytic site and mechanism representing convergence with the syn-beta-elimination site of heparinase II from Pedobacter heparinus.

Unraveling the Specificity of Heparanase Utilizing Synthetic Substrates

Heparanase is a promising anticancer target because of its involvement in cancer invasion and metastasis. Heparanase cleaves heparan sulfate (HS), a sulfated polysaccharide, and activates a series of HS-mediated cell proliferation and angiogenesis processes. Understanding the substrate specificity of heparanase will aid the discovery of heparanase inhibitors. Here, we sought to determine the specificity of heparanase using synthetic polysaccharide substrates. The substrates were prepared using purified HS biosynthetic enzymes. Using these substrates, we were able to dissect the structural moieties required for heparanase. Our data suggest that heparanase cleaves the linkage between a GlcA unit and an N-sulfo glucosamine unit carrying either a 3-O-sulfo or a 6-O-sulfo group. In addition, heparanase cleaves the linkage of a GlcA unit and N-sulfo glucosamine unit with a 2-O-sulfated GlcA residue, not a 2-O-sulfated IdoA residue, in proximity. We also discovered that the polysaccharide with repeating disaccharide units of IdoA2S-GlcNS inhibits the activity of heparanase. Our findings advance the understanding of the substrate specificity of heparanase and identify a lead compound for developing polysaccharide-based heparanase inhibitors.

Glucuronidase-assisted Transglycosylation for the Synthesis of Highly Functional Disaccharides: β-D-Glucuronyl 6-O-Sulfo-β-D-Gluco- and -β-D-Galactopyranosides

The substrate specificity of snail (Helix pomatia and Helix aspersa), limpet (Patella vulgata), and bovine glucuronidases was examined by using p-nitrophenyl glucuronide (GlcA-O-pNP) and p-nitrophenyl 6-O-sulfo-β-D-glycopyranosides as the glycosyl donor and acceptors, respectively. When the donor was treated with these enzymes in the absence of the acceptors, β (1 → 3) glucuronyl disaccharides were obtained as the major products together with β (1 → 2) isomers as the result of an enzymatic “self-transglycosylation” reaction. When p-nitrophenyl 6-O-sulfo-β-D-glucopyranosides (6-O-sulfo-Glc-O-pNP and 6-O-sulfo-Glc-S-pNP) were applied as acceptor substrates, every glucuronidase transferred the GlcA residue to either the O-3 or O-2 position in 6-O-sulfo-Glc to yield a mixture of GlcAβ (1 → 3)- and GlcAβ (1 → 2)-linked disaccharides in a ratio of 12:1 ∼ 1:1. On the other hand, when p-nitrophenyl 6-O-sulfo-β-D-galactopyranosides (6-O-sulfo-Gal-O-pNP and 6-O-sulfo-Gal-S-pNP) were applied, limpet and bovine glucuronidases gave a GlcAβ (1 → 3)-linked disaccharide regioselectively, while the snail enzymes showed no reactivity.

Antithrombin-binding Octasaccharides and Role of Extensions of the Active Pentasaccharide Sequence in the Specificity and Strength of Interaction: EVIDENCE FOR VERY HIGH AFFINITY INDUCED BY AN UNUSUAL GLUCURONIC ACID RESIDUE

The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin (AT)-binding pentasaccharide sequence AGA(*)IA (GlcN(NAc/NS,6S)-GlcA-GlcN(NS,3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA(*)IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA(*)IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (DeltaU). In two octasaccharides, AGA(*)IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA-1) or IdoUA-GlcN(NAc,6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA(*)IA was elongated at the nonreducing side by units GlcN(NS,6S)-IdoUA and GlcN(NS,6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA(*)IA, as also confirmed by fluorescence titration. Two-dimensional NMR and docking studies clearly indicated that, although elongation of the AGA(*)IA sequence does not substantially modify the bound conformation of the AGA(*)IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA(*)IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA-containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharide-AT complexes.

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by 1H-nuclear magnetic resonance spectroscopy

The relative proportion of L: -iduronic acid (IdoA) and D: -glucuronic acid (GlcA) is of great importance for the structure-function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. (1)H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the (1)H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D: -galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) (1)H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D (1)H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains.

Properties of family 79 β-glucuronidases that hydrolyze β-glucuronosyl and 4- O-methyl-β-glucuronosyl residues of arabinogalactan-protein

The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, l-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 β-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native β-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP β-GlcA occurred at pH 3.0–4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent K m values of rAnGlcAase were 30.4 μM for PNP β-GlcA and 422 μM for β-GlcA-(1→6)-Gal, and those of rNcGlcAase were 38.3 μM and 378 μM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP β-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of β-glucuronidase with the capacity to hydrolyze β-GlcA and 4-Me-β-GlcA residues of AGPs, although they differ significantly in their preferences.

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.

Structure of the Iron‐Binding Exopolysaccharide Produced Anaerobically by the Gram‐Negative Bacterium Klebsiella oxytoca BAS‐10

AbstractKlebsiella oxytoca BAS‐10 is a Gram‐negative micro‐organism capable of growing on high concentrations of heavy metals. This bacterium produces large amounts of an iron‐binding exopolysaccharide that, in the presence of metallic cations, precipitates as a dense gel. The primary and secondary structure of the repeating unit of such polysaccharide has been characterised by chemical and spectroscopic methods, resulting in the following heptasaccharide: 2)‐α‐Rha‐(1→3)‐β‐Gal‐(1→2)‐α‐Rha‐(1→4)‐β‐GlcA‐[β‐GlcA‐(1→4)]‐(1→3)‐α‐Rha‐(1→3)‐α‐Rha‐(1→. The absolute configurations for the Rha units are L, while those for the GlcA and Gal residues are D. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Chondroitin C lyase [4.2.2.] is unable to cleave fructosylated sequences inside the partially fructosylated Escherichia coli K4 polymer

Chondroitin C lyase was demonstrated to be unable to act on fructosylated sequences inside a partially fructosylated polysaccharide having the chondroitin backbone structure, the Escherichia coli K4 polymer, using different analytical approaches. Chondroitin C lyase produced various unsaturated oligosaccharides by acting on an approximately 27%-fructosylated K4 polymer. The online HPLC-ESI-MS approach showed the disaccharide nature of the main species produced by chondroitinase C as DeltaHexA-GalNAc. Furthermore, the non-digested sequences inside the K4 polymer were demonstrated to be oligosaccharides bearing a fructose for each glucuronic acid unit. In fact, unsaturated fully fructosylated oligomers, from tetrasaccharide to decasaccharide (DeltaHexA(Fru)-GalNAc-[GlcA(Fru)-GalNAc](n) with n between 1 and 4), at decreasing percentages, were produced by the enzyme. These results clearly indicate that chondroitinase C cleaved the innermost glucuronic acid-N-acetylgalactosamine linkage without affecting the 1,4 glycosidic linkage between fructosylated glucuronic acid and N-acetylgalactosamine residues, confirming that the 3-O-fructosylation of the GlcA residue renders the polysaccharide resistant to the enzyme action. This novel specific activity of chondroitinase C was also useful for the production of discrete microgram amounts of fully fructosylated oligomers, from 4- to 10-mers, from E. coli K4 for possible further studies and applications.

Mice Deficient in Heparan Sulfate 6-O-Sulfotransferase-1 Exhibit Defective Heparan Sulfate Biosynthesis, Abnormal Placentation, and Late Embryonic Lethality

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.

MS analysis of chondroitin polymerization: Effects of Mn 2+ ions on the stability of UDP-sugars and chondroitin synthesis

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl- d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn 2+ ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn 2+ ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.

Enzymatically Active N-Deacetylase/N-Sulfotransferase-2 Is Present in Liver but Does Not Contribute to Heparan Sulfate N-Sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development through interactions with growth factors and morphogens. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. NDST (glucosaminyl N-deacetylase/N-sulfotransferase), responsible for HS N-sulfation, is a key enzyme directing further modifications including O-sulfation. To elucidate the roles of the different NDST isoforms in HS biosynthesis, we took advantage of mice with targeted mutations in NDST1 and NDST2 and used liver as our model organ. Of the four NDST isoforms, only NDST1 and NDST2 transcripts were shown to be expressed in control liver. The absence of NDST1 or NDST2 in the knock-out mice did not affect transcript levels of other NDST isoforms or other HS modification enzymes. Although the sulfation level of HS synthesized in NDST1-/- mice was drastically lowered, liver HS from wild-type mice, from NDST1+/-, NDST2-/-, and NDST1+/- / NDST2-/- mice all had the same structure despite greatly reduced NDST enzyme activity (30% of control levels in NDST1+/- / NDST2-/- embryonic day 18.5 embryos). Enzymatically active NDST2 was shown to be present in similar amounts in wild-type, NDST1-/-, and NDST1+/- embryonic day 18.5 liver. Despite the substantial contribution of NDST2 to total NDST enzyme activity in embryonic day 18.5 liver (approximately 40%), its presence did not appear to affect HS structure as long as NDST1 was also present. In NDST1-/- embryonic day 18.5 liver, in contrast, NDST2 was responsible for N-sulfation of the low sulfated HS. A tentative model to explain these results is presented.

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.

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of →2)-α- d-Man(1→ and →4)-β- d-GlcA(1→, with a minor portion of →4)-β- d-Gal(1→ units. The branch points were at C-3 of →2)-Man-(1→, C-2 of →4)-Gal-(1→ and C-2 of →6)-Gal-(1→. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1→4)-, (1→3)- and (1→2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1→4)-linked. Most of the mannose units were (1→2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1→6)-linked. The absolute configurations of the sugar residues were α- d-Man p, α- l-Fuc p, α- d-Xyl p, β- d-Gal p and β- d-Glc pA. Sulfate groups in F32 were at C-6 of →2,3)-Man-(1→, C-4 and C-6 of →2)-Man-(1→, C-3 of →6)-Gal-(1→, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.

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.