An endogenous Drosophila receptor for glycans bearing alpha 1,3-linked core fucose residues

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca(2+)-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds approximately 5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing alpha1,3-linked fucose residues. Binding is enhanced by the additional presence of alpha1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.     

Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues

The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.     

Branching enzyme assay: selective quantitation of the alpha 1,6-linked glucosyl residues involved in the branching points

Methods previously described for glycogen or amylopectin branching enzymatic activity are insufficiently sensitive and not quantitative. A new, more sensitive, specific, and quantitative one was developed. It is based upon the quantitation of the glucose residues joined by alpha 1,6 bonds introduced by varying amounts of branching enzyme. The procedure involved the synthesis of a polysaccharide from Glc-1-P and phosphorylase in the presence of the sample to be tested. The branched polysaccharide was then purified and the glucoses involved in the branching points were quantitated after degradation with phosphorylase and debranching enzymes. This method appeared to be useful, not only in enzymatic activity determinations but also in the study of the structure of alpha-D-glucans when combined with those of total polysaccharide quantitation, such as iodine and phenol-sulfuric acid.     

Identification of a mung bean arabinofuranosyltransferase that transfers arabinofuranosyl residues onto (1, 5)-linked alpha-L-arabino-oligosaccharides

Arabinofuranosyltransferase activity was identified in Golgi membranes obtained from mung bean (Vigna radiata) hypocotyls. The enzyme transfers the arabinofuranosyl (Araf) residue from UDP-beta-L-arabinofuranose to exogenous (1, 5)-linked alpha-L-arabino-oligosaccharides labeled at their reducing ends with 2-aminobenzamide. The transferred residue was shown, using 1H-nuclear magnetic resonance spectroscopy and alpha-L-arabinofuranosidase treatment, to be alpha-L-Araf and to be linked to O-5 of the nonreducing terminal Araf residue of the acceptor oligosaccharide. The enzyme was nonprocessive because only a single Araf residue was added to the acceptor molecule. Arabino-oligosaccharides with a degree of polymerization between 3 and 8 were acceptor substrates. The 2-aminobenzamide-labeled arabino-tetra- and pentasaccharides were the most effective acceptor substrates analyzed. The enzyme has a pH optimum between 6.5 and 7.0 and its activity is stimulated by Mn2+ and Co2+ ions. The apparent Km and Vmax values of the arabinofuranosyltransferase for UDP-arabinofuranose are 243 microm and 243 pmol min(-1) mg protein(-1), respectively. The highest enzyme activity was detected in the elongating regions of mung bean hypocotyls. The data show that UDP-arabinofuranose is the donor molecule for the generation of arabino-oligosaccharides composed of Araf residues.     

Binding studies on internal immunodeterminants: synthesis of beta-(1----6)-linked oligosaccharide methyl glycosides having one to four internal D-galactopyranosyl residues flanked by gentiobiose residues.

The oligosaccharide glycosides beta-D-Glcp-(1----6)-beta-D-Glcp-(1----6)-[beta-D-Galp-(1----6)]n-beta-D - Glcp-(1----6)-beta-D-Glcp-1----OMe (n = 1-4) were prepared by a convergent block synthesis. Haloacetyl, tert-butyldiphenylsilyl, and dimethylthexylsilyl groups were used as temporary protective groups for the preparation of the intermediate glycosyl donors and acceptors. The deoxygenated trisaccharide glycosides beta-D-Glcp-(1----6)-beta-D-Galp-(1----6)-4-deoxy-beta-D-xylo-Hexp -1----OMe and beta-D-Glcp-(1----6)-4-deoxy-beta-D-xylo-Hexp-(1----6)-beta-D-Galp -1----OMe were also synthesized. The binding of each glycoside to the monoclonal antigalactan antibody IgA J539 was studied and the results support the previous finding that J539 can bind to internal antigenic epitopes. The data are consistent with the interpretation that subsite C of that antibody binds glucose with a Ka of approximately 6 (cf. 10.9 for galactose).     

Configurational statistics of 1,6-linked glycans

We have calculated the unperturbed dimensions of some 1,6-linked glycans by regarding the homopolymer as a ‘copolymer’ in which the different ‘comonomers’ are conformers characterized by the dihedral angle about the C5C6 bond.We find that the characteristic ratio is small (e.g. for the α-glucan it is about 3·3 to 3·4). From the results of some model calculations in which the dihedral angle about the C5C6 bond is fixed, we argue that these low values arise from bonding geometry effects, which are at least as important as the additional conformational freedom from rotation about the C5C6 bond.     

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.     

The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues.

In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by [(3)H]mannose, [(3)H]palmitic acid, and [(32)P]phosphorus. PLM has also been shown to be released from the C. albicans cell wall and to bind to and stimulate macrophage cells. In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C. albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids. We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis. Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units. NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components. Mass spectrometry analysis revealed that these chains were present in phosphoinositolmannosides with degrees of polymerization varying from 8 to 18 sugar residues. The PLM appears as a new type of eukaryotic inositol-tagged glycolipid in relationship to both the absence of glucosamine and the organization of its glycan chains. This first structural evidence for the presence of beta-1, 2-oligomannosides in a glycoconjugate other than the C. albicans phosphopeptidomannan may have some pathophysiological relevance to the adhesive, protective epitope, and signaling properties thus far established for these residues.     

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.     

H-2-linked murine factor B phenotypes

A hemolytic assay has been developed which is specific for Factor B (B) activity in murine EDTA-plasma. Three discrete levels of B activity were observed among B 10-congenic strains. Mice with standard H-2 haplotypes, b, d, k, r, f, q, s, and u, all exhibited the same mean level of activity. However, plasma from H-2 ^v (B10.SM) mice contained only 0.25 of that level, and those with standard haplotype H-2 ^ja (B10.WB) or wild haplotype H-2^wr7 (B10.WR) exhibited 2.5 times the H-2 ^b (1310) basal level of activity. These differences among B10 congenic lines suggested that the activity is H-2 controlled; further tentative mapping with intra-H-2 recombinants indicated that the gene is located in the S region. A fourth phenotype was found among progeny of backcross generations between B10.BR (H-2 ^k ) and mice of subspecies Mus musculus molossinus and M. m. bactrianus. This ultra-high activity was found also to be governed by a gene very closely linked to Ss, the primary S region marker. F₁ generations between disparate phenotypes yielded progeny with activity levels intermediate between the parents; progeny of parents of different strains with the same phenotype expressed B hemolytic titres equal to those of the parental strains. No differences in antigenic levels of the protein among the strains of different phenotypes could be detected by radial immunodiffusion. In mixing experiments, resultant activity levels were intermediate between the higher and the lower phenotype, ruling out independent inhibitors or activators of the reaction. These studies indicate that an H-2-linked S region-located single gene governs structural differences in allelic B molecules that lead to differences in specific activities.     

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).     

Gram-scale syntheses of the (1-->3)-linked and (1-->4)-linked hyaluronan disaccharides

The first gram-scale syntheses of two hyaluronan disaccharides are described. Construction of the (1-->4)-linked disaccharide 12 was achieved in 12% overall yield using 2,3-bis-dimethyl acetal protection in combination with chlorosilane-induced carbamate cleavage methodologies. The uronic acid functionality was installed using TEMPO oxidation with NaOCl as the hypochlorite source. The (1-->3)-linked disaccharide 18 was achieved in 7% overall yield utilizing acetonide protection in addition to the chlorosilane-induced carbamate cleavage methodology and the TEMPO oxidation.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.     

Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.     

Configurational statistics of single chains of α-linked glucans

Unperturbed chain conformations are evaluated assuming separable chain configuration energies. Spatial chain propagations are constructed assuming an equiprobable occurrence of conformational states within topographically selected portions of the allowed energy space. Random coil dimensions, along with spatial representations of some single chains of α-linked glucans, such as amylose, pseudo-nigerose, nigeran and linear dextran are reported. Significant architectural differences are observed for these different α-linked glucan chains, since disordered random-coil as well as persistent pseudo-helical character, either cyclic or linear, are found, depending on the type of glycosidic linkage.     

Peptides that mimic Candida albicans-derived beta-1,2-linked mannosides

Beta-1,2-linked mannosides from Candida albicans phosphopeptidomannan (PPM) bind to macrophages through a receptor independent from the macrophage alpha-linked mannose receptor and stimulate these cells to secrete immune mediators. Anti-beta-1,2-linked mannoside but not anti-alpha-linked mannoside antibodies produced after immunization with neoglycoproteins protect animals from disseminated candidiasis. In this study, peptides that mimic beta-1,2-linked mannosides were isolated using phage display methodology. A phage library expressing random peptides was panned with an anti-beta-1,2-linked mannoside monoclonal antibody (mAb). After three rounds of biopanning, the isolated phages were able to inhibit recognition of C. albicans by the mAb. Sixty percent of the phages had an identical DNA insert corresponding to the peptide sequence FHENWPS that was recognized specifically by the mAb. Injection of KLH-coupled peptide into mice generated high titers of polyclonal antibodies against C. albicans yeast cell walls. The anti-FHENWPS antibodies bound to C. albicans PPM and were inhibited by soluble beta-1,2-mannotetraose. Together, these data provide evidence for mimotopic activity of the peptide selected by biopanning with the anti-beta-1,2-oligomannoside mAb.     

Molecular mechanism underlying a Cx50-linked congenital cataract

Mutations in gapjunctional channels have been linked to certain forms of inheritedcongenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay,A. Ionides, V. Berry, A. Moore, and S. Bhattacharya.Am. J. Hum. Genet. 62: 526-532,1998). We used the Xenopus oocyte pairsystem to investigate the functional properties of a missense mutationin the human connexin 50 gene (P88S) associated with zonularpulverulent cataract. The associated phenotype for the mutation istransmitted in an autosomal dominant fashion.Xenopus oocytes injected withwild-type connexin 50 cRNA developed gap junctional conductances of~5 µS 4-7 h after pairing. In contrast, the P88S mutantconnexin failed to form functional gap junctional channels when pairedhomotypically. Moreover, the P88S mutant functioned in a dominantnegative manner as an inhibitor of human connexin 50 gap junctionalchannels when coinjected with wild-type connexin 50 cRNA. Cellsinjected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNAexhibited gap junctional coupling of ~8% and 39% of wild-typecoupling, respectively. Based on these findings, we conclude that onlyone P88S mutant subunit is necessary per gap junctional channel to abolish channel function.