Successful treatment of intraoperative myocardial ischemia with nicorandil

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.     

Structure determination for octasaccharides derived from the carbohydrate-protein linkage region of chondroitin sulphate chains in the proteoglycan aggrecan from bovine articular cartilage

Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6-year-old to 8-year-old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by beta-elimination. The individual oligosaccharides were purified by strong anion-exchange chromatography and their structures determined by very high-field one-dimensional and two-dimensional 1H-NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat-region extensions to the core tetrasaccharide linkage region structure. They have the following structures: deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc(beta1-4)+ ++GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc(beta1-4)GlcA(beta1-3)GalNAc6S(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc(b eta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol, deltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol and deltaUA(beta1-3)GalNAc4S(beta1-4)GlcA(beta1-3)GalNA c6S(beta1-4)GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl-ol. They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide-repeat-region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6-sulphated. However, in a significant amount, only one of the residues was 6-sulphated while the other was either unsulphated or 4-sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.     

Structural studies of octasaccharides derived from the low-sulfated repeating disaccharide region and octasaccharide serines derived from the protein linkage region of porcine intestinal heparin

Four octasaccharide serines and three octasaccharides were isolated after heparinase treatment of porcine intestinal heparin. Their structures were characterized by enzymatic digestion in conjunction with HPLC and 500 MHz 1H NMR spectroscopy. Three of the four octasaccharide serines were structurally identical with those isolated previously, whereas one has the unreported structure DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4GlcAbe ta1-4GlcNAca lpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta 1-O-Ser (DeltaHexA, GlcN, IdceA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, respectively). The other three octasaccharides were isolated for the first time as discrete structures and shared the common core hexasulfated sequence DeltaHexA(2-sulfate)alpha1-4GlcN(N-sulfate)alpha1-4IdceAa lpha1-4GlcNA calpha1-4GlcAbeta1-4GlcN(N-sulfate)alpha1-4IdceA (2-sulfate)alpha1-4Gl cN(N,6-disulfate) with one or two additional sulfate groups. The octasaccharides which were derived from the low-sulfated repeating disaccharide region of heparin contained the common trisaccharide sequence -4IdceAalpha1-4GlcNAcalpha1-4GlcAbeta1- [Yamada, S., Yamane, Y., Tsuda, H., Yoshida, K., and Sugahara, K. (1998) J. Biol. Chem. 273, 1863-1871], suggesting the programmed biosynthesis of heparin. These octasaccharides are the largest oligosaccharides isolated so far from the low-sulfated irregular region of heparin. Since oligosaccharides larger than a pentasaccharide appear to potentially exhibit binding activities toward growth factors or other functional proteins, they will be useful for investigating the structural requirement for molecular interactions between heparin and/or heparan sulfate and biologically active proteins. During the course of the present structural studies, we evaluated the NMR data accumulated thus far on heparin oligosaccharides and found several interesting rules on chemical shifts of proton signals affected by the neighboring sugar residues and their sulfation, which will be in turn useful for determining structures of unknown heparin and/or heparan sulfate oligosaccharides based on the proton resonances.     

Biosynthesis of the escherichia coli K4 capsule polysaccharide. A parallel system for studies of glycosyltransferases in chondroitin formation

Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin. GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-[14C]GlcA and UDP-[3H]GalNAc, respectively. It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases. The radiolabeled products were completely degraded with chondroitinase AC; the [14C]GlcA unit could be removed by beta-D-glucuronidase, and the [3H]GalNAc could be removed by beta-N-acetylhexosaminidase. A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive. These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer. This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis. In the biosynthesis of capsule polysaccharides from E. coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 255, 87-101). In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P. (1983) Biochem. J. 211, 181-189).     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.     

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.     

Variations in the chondroitin sulfate-protein linkage region of aggrecans from bovine nasal and human articular cartilages

Aggrecan-derived chondroitin sulfate (CS) chains, released by beta-elimination, were derivatized with p-aminobenzoic acid or p-aminophenol; radioiodinated; and subjected to graded or complete degradations by chondroitin ABC lyase to generate linkage region fragments of the basic structure DeltaGlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (where DeltaGlyUA represents 4, 5-unsaturated glycuronic acid, and R is the adduct), by chondroitin AC lyase to generate the shorter fragment DeltaGlyUA-Gal-Gal-Xyl-R, or by chondroitin C lyase to generate the same fragment when it was linked to a 6-O-sulfated or unsulfated GalNAc at the nonreducing end. Fragments were separated by size using gel chromatography, by charge using ion-exchange chromatography, and by size/charge using electrophoresis and then characterized by stepwise degradations from the nonreducing end by using mercuric acetate to remove all terminal DeltaGlyUA, by bacterial glycuronidase to remove the same residue when linked to unsulfated or 6-O-sulfated GalNAc/Gal, by mammalian 4-sulfatase to remove sulfate from terminal GalNAc 4-O-sulfate, by chondro-4-sulfatase to remove 4-O-sulfate from other GalNAc/Gal residues, and by beta-galactosidase to remove terminal Gal. Results with CS from bovine nasal cartilage aggrecan show that, in nearly all chains, Xyl and probably also the first Gal are unsubstituted, whereas the second Gal is 4-O-sulfated in one CS chain out of five. The first disaccharide repeat is sulfated at C-4 of GalNAc in one chain out of three and unsulfated in the other two. A sulfated first disaccharide is always joined to an unsulfated GlcUA-Gal-Gal sequence. In contrast, CS from human articular cartilage usually has a sulfated first disaccharide repeat. In CS from young human cartilage, sulfate groups are mostly at C-4 of GalNAc in the major part of the chain, but at C-6 in the nonreducing distal portion. In CS from old cartilage, sulfation at C-6 of GalNAc is a major feature from the nonreducing end down to approximately positions 4 and 5 from the linkage region, where GalNAc 4-O-sulfate is common.     

Changes in glycosaminoglycan characteristics during progression of a human gingival carcinoma xenograft line in nude mice

We investigated changes in the glycosaminoglycans (GAGs) during progression of a human gingival carcinoma xenograft line, GK -1, in nude mice. The GAGs extracted from cancers 3, 5, 7, 10 and 15 weeks after transplantation consisted of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS) as major components, and dermatan sulfate (DS) as a trace component for all cancers. HPLC analysis revealed that the HA content per defatted tissue dry weight increased in the cancers 5 weeks after transplantation compared to those of 3 weeks (p < 0.05), while CS for cancers at 10 weeks decreased compared with 7 weeks (p < 0.05). However, HS showed no significant change. Both the CS and DS contained primarily 4-sulfated disaccharide units. Immunohistochemical staining with antibody 2-B-6 for the PGs having delta DI-4S produced by chondroitinase ABC digestion showed that CS is located in the tissue surrounding the cancer nests and mass. These results indicate that the location of accumulation of CS, which primarily contains 4-sulfated disaccharide units, plays an important role in cancer progression.     

Synthesis of oligosaccharides related to HNK-1 antigen. 2. Synthesis of 3'-O-(3-O-sulfo-beta-D-glucuronopyranosyl)-lacto-N-neotetrao se beta-propylglycoside

A derivative of allyl 3"-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-lactoside with a free OH group at C-4GlcNAc was glycosylated with trichloroacetimidate of selectively protected GlcA(beta 1-->3)Gal alpha disaccharide in dichloromethane in the presence of trimethylsilyl triflate resulting in a pentasaccharide product with an 82% yield. This product was converted to monohydroxy derivative with a free OH group at C-3GlcA via the formation and the subsequent opening of the 6,3-lactone ring in the glucuronic acid residue. The 3"'-O-sulfation of the monohydroxy derivative, the removal of the protective groups, and the reduction of the allyl aglycon yielded the pentasaccharide propyl glycoside NaSO3-3GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-Opr comprising the oligosaccharide chain of the SGGL-1 glycolipid, which is recognized by HNK-1 antibodies. NaSO3-3GlcA(beta 1--> 3)Gal beta OAll, GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-OPr and GlcA(beta 1-->3)Gal beta OAll were synthesized in a similar way.     

Acute osteo-articular infection of the limbs of children. Physiopathology, diagnosis, progression, prognosis, treatment

Chondroitin sulfates were isolated from the mud snail. For the quantitative analysis of enzymatic digestion products of isolated chondroitin sulfates, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitinase ABC, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS), 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S) and 2-acetamide-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S) were produced from the mud snail chondroitin sulfates. The analysis showed that relative proportion of delta Di-OS/delta Di-6S/delta Di-4S was 58.7/3.1/38.2. The immunomodulating activity of chondroitin sulfate was examined by cell proliferation assay and these results suggest that it might be a immunosuppressant.     

Characterization of heparin and heparan sulfate domains binding to the long splice variant of platelet-derived growth factor A chain

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.     

Brain-specific receptor-type protein-tyrosine phosphatase RPTP beta is a chondroitin sulfate proteoglycan in vivo

By using polyclonal antiserum, which recognizes multiple proteoglycan core proteins, we isolated a cDNA species for an unknown chondroitin sulfate proteoglycan in bovine brain. Unexpectedly, DNA sequencing revealed that the cDNA encodes an open reading frame highly homologous to the human receptor-type protein-tyrosine phosphatase, RPTP beta. To prove that RPTP beta is a proteoglycan, we raised three polyclonal antibodies against extracellular and cytoplasmic domains of human RPTP beta. These antibodies have been shown to react with a smear band ranging from 350 to 500 kDa in human brain extracts. Digestion with chondroitinase ABC eliminated this smear and gave rise to a 310/300-kDa doublet band that was not detected without digestion, indicating that almost all of the RPTP beta molecules in the brain contain chondroitin sulfate chains. In the cerebellum, immunofluorescence staining of chondroitinase-treated sections revealed pericellular localization of RPTP beta in the external and internal granular layers. These data establish that RPTP beta is expressed constitutively as a chondroitin sulfate proteoglycan in the brain, and suggest that chondroitin sulfates may be an essential component for the physiological function of RPTP beta in vivo.     

Enzymatic synthesis of N-linked oligosaccharides terminating in multiple sialyl-Lewis(x) and GalNAc-Lewis(x) determinants: clustered glycosides for studying selectin interactions

Galactosyltransferase, sialyltransferase, and fucosyltransferase were used to create a panel of complex oligosaccharides that possess multiple terminal sialyl-Le(x) (NeuAc alpha 2-3Gal[Fuc alpha 1-3] beta 1-4GlcNAc) and GalNAc-Le(x) (GalNAc[Fuc alpha 1-3]beta 1-4GlcNAc). The enzymatic synthesis of tyrosinamide biantennary, triantennary, and tetraantennary N-linked oligosaccharides bearing multiple terminal sialyl-Le(x) was accomplished on the 0.5 mumol scale and the purified products were characterized by electrospray MS and 1H NMR. Likewise, biantennary and triantennary tyrosinamide oligosaccharides bearing multiple terminal GalNAc-Le(x) determinants were synthesized and similarly characterized. The transfer kinetics of human milk alpha 3/4-fucosyltransferase were compared for biantennary oligosaccharide acceptor substrates possessing Gal beta 1-4GlcNAc, GalNAc beta 1-4GlcNAc, and NeuAc alpha 2-3Gal beta 1-4GlcNAc which established NeuAc alpha 2-3Gal beta 1-4GlcNAc as the most efficient acceptor substrate. The resulting complex oligosaccharides were chemically tethered through the tyrosinamide aglycone to the surface of liposomes containing phosphatidylthioethanol, resulting in the generation of glycoliposomes probe which will be useful to study relationships between binding affinity and the micro- and macro-clustering of selectin ligand.     

Enzymatic synthesis of oligosaccharides containing Gal beta-->4Gal disaccharide at the non-reducing end using beta-galactanase from Penicillium citrinum

The transglycosylation reaction was done with a beta-galactanase from Penicillium citrinum. The regioselectivity in the transglycosylation reaction was studied using soy bean arabinogalactan as a donor and mono- or disaccharide derivatives containing beta-galactosyl residue as acceptors. We also synthesized oligosaccharides containing Gal beta 1-->4Gal sequence such as Gal beta 1-->4Gal beta1-->4Glc, Gal beta 1-->4Gal beta 1-->3GlcNac, Gal beta 1-->4Gal beta 1-->4GlcNAc, Gal beta 1-->4Gal beta 1-->6GlcNAc, and Gal beta 1-->4Gal beta 1-->3GalNAc for use in the total synthesis of complex sugar chains.     

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.     

Molecular species analysis of glycosphingolipids from small intestine of Japanese quail, Coturnix coturnix Japonica by HPLC/FAB/MS

Neutral glycosphingolipids were isolated from quail small intestine and their structures were analysed. They contained: Gal beta 1-4GlcCer(LacCer), Gal alpha 1-4GalCer(Ga2Cer), Gal alpha 1-4Gal beta 1-4GlcCer(Gb3Cer), GlcNAc beta 1-3Gal beta 1-4GlcCer(Lc3Cer), GalNAc beta 1-4Gal beta 1-4GlcCer(Gg3Cer), GalNAc beta 1-4[GalNAc beta 1-3] Gal beta 1-4GlcCer(LcGg4Cer), and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4GlcCer (Forssman glycolipid) as well as glucosylceramide, galactosylceramide (Nishimura K et al. 1984) Biochim Biophys Acta 796:269-76) and the LeX glycolipid, III3 Fuc alpha-nLc4Cer (Nishimura K et al. (1989) J. Biochem (Tokyo) 101:1315-18). The molecular species compositions of these glycosphingolipids were examined using fast atom bombardment-mass spectrometry linked with reversed-phase high-performance liquid chromatography. By such analysis, we could classify the quail glycosphingolipids into at least three classes: glycolipids rich in species having four hydroxyl groups in the ceramides (GalCer, Gg3Cer, LcGg4Cer and LeX), those rich in the ceramides of N-acyl trihydroxysphinganine with normal fatty acids (Lc3Cer), and glycolipids rich in the ceramides of N-acyl sphingenine with normal fatty acids (LacCer, Gb3Cer and Forssman glycolipid). Immunohistochemical observation implies that the differences in the hydrophobic moieties specified the localization of glycosphingolipids in the tissue.     

Differential release of proteoglycans during human B lymphocyte maturation

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

6B4 proteoglycan/phosphacan, an extracellular variant of receptor-like protein-tyrosine phosphatase zeta/RPTPbeta, binds pleiotrophin/heparin-binding growth-associated molecule (HB-GAM)

A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta/RPTPbeta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (Kd = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 microg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPzeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.