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PubMed1970

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls.

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.

Bacillus↗
PubMed1971

Studies on the carbohydrate moiety of 1 -acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid.

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.

Alkalies↗
PubMed1992

Structural differences between heparan sulphates of proteoglycan involved in the formation of basement membranes in vivo by Lewis-lung-carcinoma-derived cloned cells with different metastatic potentials.

This study addresses the characterization of heparan sulphates of the basement-membrane proteoglycans in tumour formed after the subcutaneous implantation of Lewis-lung-carcinoma-derived different metastatic clones (P29, LM12-3 and LM60-D6 clones with low, medium and high metastatic potentials respectively). Heparan sulphate proteoglycans (125-158 micrograms of hexuronate/g dry weight of tissue) were isolated from chondroitin ABC lyase digests of a proteoglycan fraction obtained after DEAE-Sephacel chromatography of tissue extracts. The proteoglycans were separated into three molecular species by Sepharose CL-4B chromatography followed by CsCl-density-gradient centrifugation: large proteoglycans with an estimated M(r) of 820,000-130,000, which consisted of two components with low (< 1.34 g/ml; PGII-M) and high (> 1.37 g/ml; PGII-B) density, and a small proteoglycan with an M(r) of less than 80,000 (PGIII). Of these, only the PGII-M proteoglycan (34-37 micrograms of hexuronate/g dry weight) reacted with the antiserum against proteoglycan of Engelbreth-Holm-Swarm-tumour basement membrane, and represented, therefore, a basement-membrane proteoglycan. Digestion with heparan sulphate lyases I and II of the heparan sulphates (M(r) 36,000) from the PGII-M proteoglycan of the three tumours resulted in almost complete depolymerization to give six unsaturated disaccharides identified as 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-D-glucose, 2-acetamido-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyranosyluron ic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-alpha-L-threo-hex-4-enopyrano syluronic acid)-6-O-sulpho-D-glucose, 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-D-glucose and 2-deoxy-2-sulphamino-4-O-(4-deoxy-2-O-sulpho-alpha-L-threo-hex-4- enopyranosyluronic acid)-6-O-sulpho-D-glucose. Comparison of the relative amounts of these disaccharides produced from the three tumour-derived heparan sulphates demonstrated that the degree of sulphation of the heparan sulphates correlated with the degree of morphological organization of the tumour basement membranes; the heparan sulphate from the more highly metastatic tumour with more highly organized basement membrane exhibited a higher degree of overall sulphation along the glycosaminoglycan chains, which was due to an increased content of the three repeating disaccharides having 6-O-sulphated glucosamine residues.

Animals↗
PubMed1995

Heparin proteoglycans synthesized by mouse mastocytoma contain chondroitin sulphate.

Proteoglycans (PGs), biosynthetically labelled with [35S]sulphate, were isolated from mouse mastocytoma tissue. Chromatography on antithrombin (AT)-Sepharose resulted in the separation of the 35S-labelled PGs into three fractions: PGs with no affinity for the gel (NA-PGs), PGs with low affinity (LA-PGs), and PGs with high affinity (HA-PGs) for antithrombin. Whereas NA-PGs contained almost exclusively chondroitin sulphate (CS), the AT-binding PGs contained 80-85% heparin and 15-20% CS. [35S]CS-containing macromolecules obtained from the HA-PG fraction after removal of the heparin polysaccharide chains were rechromatographed on AT-Sepharose. A majority of these 35S-labelled macromolecules no longer showed affinity for AT. These experiments indicate that the [35S]CS recovered in the AT-binding PGs is present in hybrid PGs. Polysaccharide chain-length determination demonstrated that the heparin chains were somewhat larger (M(r) approximately 30,000) than the CS chains in the NA-PGs (M(r) approximately 25,000). CS chains in the hybrid PGs were slightly smaller (M(r) approximately 20,000). Characterization of the sulphated CS disaccharides from NA- and HA-PGs showed that they contained similar amounts (20%) of disulphated disaccharides of [GlcA-GalNAc(4,6-di-OSO3)] type. The monosulphated CS-disaccharides were O-sulphated at C-4 of the galactosamine units. Analysis by gel chromatography of the [35S]CS components isolated from HA-PGs after heparinase treatment showed that a major portion of these contained one CS chain only. Calculations of the number of CS and heparin chains in AT-binding PGs, based on polysaccharide composition and polysaccharide chain length, indicate that all heparin-containing PGs are hybrids.

Animals↗
PubMed1998

Structural characterisation of two hexasaccharides and an octasaccharide from chondroitin sulphate C containing the unusual sequence (4-sulpho)-N-acetylgalactosamine-beta1-4-(2-sulpho)-glucuronic acid-beta1-3-(6-sulpho)-N-acetylgalactosamine.

The structures of two hexasaccharides and an octasaccharide, isolated from shark cartilage chondroitin sulphate C after partial chondroitin ABC lyase digestion, have been elucidated by disaccharide analysis, liquid secondary-ion mass spectrometry, and 1H/13C NMR spectroscopy. In the case of the octasaccharide, the order of the internal disaccharide units was deduced from a novel one-dimensional TOCSY/ROESY excitation sculpting experiment [Gradwell M. J., Kogelberg, H. & Frenkiel, T. A. (1997) J. Magn. Reson. 124, 267-270] which incorporates two double-pulsed field-gradient spin-echo sequences. The oligosaccharides each contain the unusual motif GalNAc(4SO3-)beta1-4GlcA(2SO3-)beta1-3GalNAc(6SO3++ +-). From the adjoining sequences in the oligosaccharides, it is apparent that this motif occurs interspersed with typical chondroitin sulphate-C disaccharides, GlcAbeta1-3GalNAc(6SO3-). Such differentially sulphated domains along the glycosaminoglycan chains are potential sites for specific recognition processes.

Carbohydrate Sequence↗
PubMed1994

Modulation by transforming growth factor-beta 1 and interleukin-1 beta of proteoglycan release and chondrodisaccharide composition in porcine articular cartilage.

The effects of IL-1 beta and TGF-beta 1 on proteoglycan release and modifications in porcine articular cartilage explants are described. Proteoglycan release in culture medium was markedly increased when cartilage was treated with IL-1 beta (5 to 100 ng/mL). Addition of IL-1 beta alone or in combination with TGF-beta 1 to cartilage culture stimulated the production and release of PGE2. Indomethacin (10(-6) M) did not modify the IL-1 beta effect on proteoglycan release. Both the spontaneous and IL-1 beta-induced proteoglycan release were downregulated by TGF-beta 1 (50 ng/mL). Basal and stimulated loss of proteoglycans was reduced by the addition of cycloheximide (10(-6) M) and by metalloprotease inhibitors. GAGs of conditioned medium and extracellular matrix proteoglycans were digested to about 90% by chondroitin ABC lyase, suggesting that CS is the major GAG present. Structural analysis of disaccharides, by CZE, revealed a different pattern of oxysulfation when cartilage explants were treated either with IL-1 beta or TGF-beta 1. Indeed, analysis of GAGs, released into the medium from TGF-beta 1-treated cartilage, showed a reduction in the level of 4-S-disaccharide (delta Di4S and an increase in desulfated disaccharides delta Di0S). In the extracellular matrix, IL-1 beta and TGF-beta 1 induced a more complex rearrangement of the GAGs, in that the levels of delta Di0S and delta Di4S were increased whereas those of delta Di6S were decreased. Altogether, these results suggest that TGF-beta 1 does not only counteract the effect of IL-1 beta on proteoglycan release, but modifies the structure of GAGs, probably by interfering with the activity or the synthesis of sulfotranspherases involved in GAG biosynthesis.

Animals↗
PubMed1998

Molecular characterization of Xenopus embryo heparan sulfate. Differential structural requirements for the specific binding to basic fibroblast growth factor and follistatin.

Enzymatic elimination of heparan sulfate (HS) causes abnormal mesodermal and neural formation in Xenopus embryos, and HS plays an indispensable role in establishing the embryogenesis and tissue morphogenesis during early Xenopus development (Furuya, S., Sera, M., Tohno-oka, R., Sugahara, K., Shiokawa, K., and Hirabayashi, Y. (1995) Dev. Growth Differ. 37, 337-346). In this study, HS was purified from Xenopus embryos to investigate its disaccharide composition and binding ability to basic fibroblast growth factor (bFGF) and follistatin (FS), the latter being provided in two isoforms with core sequences of 315 and 288 amino acids (designated FS-315 and FS-288) originating from alternative mRNA splicing. Disaccharide composition analysis of the purified Xenopus HS showed the preponderance of a disulfated disaccharide unit with uronic acid 2-O-sulfate and glucosamine 2-N-sulfate, which has been implicated in the interactions with bFGF. Specific binding of the HS to bFGF and FS-288, the COOH-terminal truncated form, was observed in the filter binding assay, whereas HS did not bind to FS-315, indicating that the acidic Glu-rich domain of FS-315 precluded the binding. The binding of the HS to bFGF or FS-288 was markedly inhibited by heparin (HP) and various HS preparations, but not by chondroitin sulfate, supporting the binding specificity of HS. The binding specificity was further investigated using FS-288 and bovine intestinal [3H]HS. Competitive inhibition assays of the HS binding to FS-288 using size-defined HP oligosaccharides revealed that the minimum size required for significant inhibition was a dodecasaccharide, which is larger than the pentasaccharide required for bFGF binding. The binding affinity of FS to HS increased in the presence of activin, a growth/differentiation factor, which could be inactivated by direct binding to FS. These results, taken together, indicate that the structural requirement for binding of HS to bFGF and FS is different. HS may undergo dynamic changes in its structure during early Xenopus embryogenesis in response to the temporal and spatial expression of various growth/differentiation factors.

Activins↗
PubMed2001

Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence.

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.

Animals↗
PubMed2003

The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. Molecular cloning, recombinant expression, and biochemical characterization.

Heparan sulfate glycosaminoglycans are structurally complex polysaccharides critically engaged in a wide range of cell and tissue functions. Any structure-based approach to study their respective biological functions is facilitated by the use of select heparan sulfate glycosaminoglycan-degrading enzymes with unique substrate specificities. We recently reported of one such enzyme, the Delta4,5-glycuronidase cloned from Flavobacterium heparinum and recombinantly expressed in Escherichia coli (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41, 7424-7434). In this study, we likewise report the molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active enzyme. At the protein level, the flavobacterial 2-O-sulfatase possesses considerable sequence homology to other members of a large sulfatase family, especially within its amino terminus, where the highly conserved sulfatase domain is located. Within this domain, we have identified by sequence homology the critical active site cysteine predicted to be chemically modified as a formylglycine in vivo. We also present a characterization of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of its substrate specificity. In particular, we demonstrate that in addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate. The sulfatase also displays a clear kinetic preference for disaccharides with beta1-->4 linkages but is able, nevertheless, to hydrolyze unsaturated, 2-O-sulfated chondroitin disaccharides. Finally, we describe the substrate-product relationship of the 2-O-sulfatase to the Delta4,5-glycuronidase and the analytical value of using both of these enzymes in tandem for elucidating heparin/heparan sulfate composition.

Amino Acid Sequence↗
PubMed2004

The structure of chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent catalytic machinery.

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

Amino Acid Sequence↗
PubMed2005

Heparin-binding growth factor, pleiotrophin, mediates neuritogenic activity of embryonic pig brain-derived chondroitin sulfate/dermatan sulfate hybrid chains.

Chondroitin sulfate (CS) and dermatan sulfate (DS) chains play roles in the central nervous system. Most notably, CS/DS hybrid chains (E-CS/DS) purified from embryonic pig brains bind growth factors and promote neurite outgrowth toward embryonic mouse hippocampal neurons in culture. However, the neuritogenic mechanism is not well understood. Here we showed that pleiotrophin (PTN), a heparin-binding growth factor, produced mainly by glia cells, was the predominant binding partner for E-CS/DS in the membrane-associated protein fraction of neonatal rat brain. The CS/DS chains were separated on a PTN column into unbound, low affinity, and high affinity fractions. The latter two fractions promoted outgrowth of dendrite- and axon-like neurites, respectively, whereas the unbound fraction showed no such activity. The activity of the low affinity fraction was abolished by an anti-PTN antibody or when glia cells were removed from the culture. In contrast, the high affinity fraction displayed activity under both these conditions. Hence, PTN mainly from glia cells mediated the activity of the low affinity but not the high affinity fraction. The anti-CS antibody 473HD neutralized the neuritogenic activities of both fractions. Interaction analysis indicated that the 473HD epitope and PTN-binding domains in the E-CS/DS chains largely overlap. The three affinity subfractions differed in disaccharide composition and the distribution of l-iduronic acid-containing disaccharides along the chains. Oversulfated disaccharides and nonconsecutive iduronic acid-containing units were the requirements for the E-CS/DS chains to bind PTN and to exhibit the neuritogenic activities. Thus, CS subpopulations with distinct structures in the mammalian brain play different roles in neuritogenesis through distinct molecular mechanisms, at least in part by regulating the functions of growth factors.

Amino Acid Sequence↗
PubMed1998

Beneficial effect of carbohydrate maldigestion induced by a disaccharidase inhibitor (AO-128) in the treatment of chronic portal-systemic encephalopathy. A double-blind, randomized, controlled trial.

BACKGROUND: The most widely used treatment of portal-systemic encephalopathy (PSE) is the administration of oral, non-absorbable disaccharides. Theoretically, the inhibition of intestinal disaccharidases should induce malabsorption of disaccharides and increase delivery of undigested carbohydrates to the colon, thus stimulating the effects of lactulose and other non-absorbable disaccharides (that is, lactitol and lactose). AO-128 is an N-substituted derivative of valeolamine, an aminocyclitol that selectively inhibits intestinal disaccharidases. This study was performed to investigate whether AO-128 could be used as adjuvant therapy for the treatment of mild PSE in cirrhotic patients. METHODS: A double-blind, randomized, controlled trial was performed in 35 cirrhotic patients with PSE. Patients were given a 2-week treatment consisting of AO-128 (2 mg three times daily) or an identical placebo. The following features of PSE syndrome were assessed in a semiquantitative fashion before and after I and 2 weeks of therapy: mental state, asterixis, number connection test (NCT), venous blood ammonia concentration, electroencephalogram (EEG), and overall PSE index (PSEI). More patients receiving AO-128 than patients receiving placebo showed >40% improvement in the PSEI (83% versus 35%; P < 0.05). The mean stool pH decreased from 5.8+/-0.3 to 5.5+/-0.3 (P < 0.004) after AO-128 treatment, whereas no changes were observed in the placebo group. The EEG and nitrogen balance did not show significant changes in any of the two groups. A significant improvement was seen in the NCT performance after AO-128 (from grade 2.0+/-1.04 to grade 1.25+/-0.87; P < 0.05). Seven patients treated with AO-128 developed diarrhea, as compared with none in the placebo group (P < 0.05). CONCLUSION: These results suggest that AO-128 may be useful in the treatment of PSE, although further studies are required to establish the benefit of AO-128 and determine adequate individual doses.

Cyclohexanols↗
PubMed2000

Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE).

Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Deltadisaccharides including DeltaDiHA, DeltaDi0S, DeltaDi2S, DeltaDi4S, DeltaDi6S, DeltaDi2,4S, DeltaDi2,6S, DeltaDi4,6S, and DeltaDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4, 6S-galNAc.

Acetylgalactosamine↗
PubMed2006

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core.

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.

Animals↗
PubMed2000

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase.

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.

Animals↗
PubMed1995

A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins.

The uptake of monosaccharides (glucose and xylose) and disaccharides (cellobiose and xylobiose) was evaluated in the Streptomyces lividans mutant strain 10-164. The pleiotropic mutation had no effect on glucose uptake; however, the Vmax of xylose uptake was decreased 10-fold as compared to the wild-type strain, S. lividans 1326, and the transport system of cellobiose and xylobiose, the putative inducers of the cellulase and xylanase genes, was completely abolished resulting in a cellulase/xylanase-negative mutant. An accumulation of xylose and glucose in culture media was observed when the mutant was grown on xylobiose and cellobiose, respectively. Cell-associated beta-glucosidase and low levels of extracellular beta-glucosidase were detected in both strains. When gluconolactone, a beta-glucosidase inhibitor, was added to the medium there was no uptake of cellobiose or release of glucose by the mutant strain, whereas the uptake of cellobiose by the wild-type strain was not significantly affected. It is thus proposed that the active transport system for cellobiose and xylobiose is affected in mutant strain 10-164. Glucose and xylose production from disaccharide hydrolysis are due to beta-glucosidase and beta-xylosidase activities, which sustain the growth of the mutant strain. Clones complementing the mutation were isolated from a gene bank constructed using mutant strain 10-164. The msiK gene codes for MsiK, a 40 kDa multiple sugar import protein, which belongs to the family of ATP-binding proteins. The mutation is located in the B site which is responsible for ATP binding. This protein probably provides energy to the xylose and disaccharide transport system as a result of the hydrolysis of ATP.

Adenosine Triphosphatases↗
PubMed2004

Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B isolectin to alphagalactosyl carbohydrate antigens in the surface phase.

The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.

Agaricales↗
PubMed1977

Investigation into the occurrence and structure of lectin receptors on human and bovine erythrocytes milk fat globule and lymphocyte plasma-membrane glycoproteins.

The reduced disaccharide beta-D-galactosyl(1-3)-N-acetyl-D-galactosaminitol was released, after alkaline borohydride and neuraminidase treatment, from human and bovine erythrocyte membrane and fat globule membrane glycoproteins as well as from human and bovine lymphocyte plasma membranes. This disaccharide was found to be substituted to different extents by sialic acid in the various fractions. beta-D-Galactosyl(1-3)-N-acetyl-D-galactosamine is the immunodominant disaccharide of the Thomsen-Friedenreich antigen (TF) and was detected serologically using specific lectins and antibody-containing sera. Bovine fat globule membrane glycoproteins were the only source of Tn antigen. The Tn antigen was detected by anti-Tn-containing sera and by the lectins from Salvia sclarea and Helix pomatiea as well as gas chromatographic detection of N-acetylgalactosaminitol after alkaline borohydride treatment of the native glycoproteins. Human milk fat globule membrane glycoproteins showed the presence of blood group A substance by reaction with rabbit anti-A serum.

ABO Blood-Group System↗