Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase

The chemoenzymatic synthesis of a glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide is described. We synthesized glycosylated Peptide T which blocks infection of human T cells by human immunodeficiency virus. The first step of the chemoenzymatic method is the solid-phase chemical synthesis of N-acetylglucosaminyl Peptide T (Ala-Ser-Thr-Thr-Thr-Asn(GlcNAc)-Tyr-Thr) with an N-acetylglucosamine moiety bound to the asparaginyl residue by a solid-phase method. This product was prepared in high yield by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety using Fmoc-N-acetylglucosaminyl asparagine instead of Fmoc-asparagine. The second step was transglycosylation of complex type oligosaccharide to N-acetylglucosaminyl Peptide T by a microbial endoglycosidase. The endo-beta-N-acetylglucosaminidase of Mucor hiemalis transfer the oligosaccharide of human transferrin glycopeptide to N-acetylglucosaminyl Peptide T. The transglycosylation product was confirmed to be the glycosylated Peptide T with a sialo biantennary complex type oligosaccharide by mass spectrometry. The glycosylated Peptide T was highly stable against proteolysis in comparison to native Peptide T and N-acetylglucosaminyl Peptide T.     

Chemoenzymatic synthesis of chiral biologically active heterocycles

During 1995, among 1105 HIV patients explored in our department, 64 presented a deep fungic infection (5.8%). The yeast was searched for in cerebrospinal fluid, blood, urine, and bronchoalveolar aspiration. Isolated germs were Cryptococcus neoformans (95%), Candida tropicalis (1 case), Saccharomyces cerevisiae (1 case) et Aspergillus fumigatus (1 case). Results of treatment with amphotericin B were: recovery (9%), clinical success (11%), out of sight (14%), letality (66%), relapse (23%) and side effects (19%). We emphasized diagnostical and therapeutical difficulties, and bad prognostic of mycoses in patients with AIDS.     

The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion

Large-scale enzymatic synthesis of oligosaccharides, which contain terminal N-acetyl-neuraminic acid residues requires large amounts of the sialyltransferase and the corresponding sugar-nucleotide synthetase, which is required for the synthesis of the sugar-nucleotide donor, CMP-Neu5Ac. Using genes cloned from Neisseria meningitidis, we constructed a fusion protein that has both CMP-Neu5Ac synthetase and alpha-2,3-sialyltransferase activities. The fusion protein was produced in high yields (over 1200 U/L, measured using an alpha-2,3-sialyltransferase assay) in Escherichia coli and functionally pure enzyme could be obtained using a simple protocol. In small-scale enzymatic syntheses, the fusion protein could sialylate various oligosaccharide acceptors (branched and linear) with N-acetyl-neuraminic acid as well as N-glycolyl- and N-propionyl-neuraminic acid in high conversion yield. The fusion protein was also used to produce alpha-2,3-sialyllactose at the 100 g scale using a sugar nucleotide cycle reaction, starting from lactose, sialic acid, phosphoenolpyruvate, and catalytic amounts of ATP and CMP.     

Chemoenzymatic synthesis of classical and non-classical anticoagulant heparan sulfate polysaccharides

Heparan sulfate (HS) polysaccharides interact with numerous proteins at the cell surface and orchestrate many different biological functions. Though many functions of HS are well established, only a few specific structures can be attributed to HS functions. The extreme diversity of HS makes chemical synthesis of specific bioactive HS structures a cumbersome and tedious undertaking that requires laborious and careful functional group manipulations. Now that many of the enzymes involved in HS biosynthesis are characterized, we show in this study how one can rapidly and easily assemble bioactive HS structures with a set of cloned enzymes. We have demonstrated the feasibility of this new approach to rapidly assemble antithrombin III-binding classical and non-classical anticoagulant polysaccharide structures for the first time.     

Chemoenzymatic synthesis of the branched oligosaccharides which correspond to the core structures of N-linked sugar chains

Synthetic routes are described to a partial structure common to all high mannose-type sugar chains and complex-type sugar chains based on a chemoenzymatic strategy which incorporates, (a) enzymatic synthesis of oligosaccharide blocks using glycosidases, and (b) chemical synthesis of the branching oligosaccharides via regioselective coupling. All reaction products correspond to key intermediates necessary for the construction of N-linked oligosaccharides and we have synthesized the branched tetra-manno-oligosaccharide high mannose-type sugar chain and the branched hexa-oligosaccharide complex-type sugar chain using this simple and direct method.     

Chemoenzymatic synthesis of iminocyclitol derivatives: a useful library strategy for the development of selective fucosyltransfer enzymes inhibitors

A chemoenzymatic strategy has been developed for the synthesis of libraries of iminocyclitol derivatives for the discovery of new and selective fucosidase inhibitors.     

Chemoenzymatic synthesis of a 3IV,6III-disulfated Lewis(x) pentasaccharide, a candidate ligand for human L-selectin

The disulfated pentasaccharide 3-O-SO3(-)-beta-D-Galp-(1-->4)-[alpha-L-Fucp-(1-->3)]-6-O-SO3(-)- beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp was prepared according to a chemoenzymatic approach, starting from 4-methoxybenzyl O-(4-O-acetyl-2,6-di-O-benzyl-beta- D-galactopyranosyl)-(1-->4)-O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, obtained in six steps from hepta-O-acetyl lactosyl bromide. Coupling of this lactose derivative with O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) trichloracetimidate afforded, after dephthaloylation and re-N-acetylation, 4-methoxybenzyl O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1-->3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-- >4)- O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Regioselective sulfation at the primary position of the glucosamine residue was then successfully achieved and the benzyl groups were removed. Enzymatic galactosylation of 4-methoxybenzyl O-(2-acetamido-2-deoxy-6-O-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl-(1-->4)-O-beta-D- glucopyranoside sodium salt, and subsequent regioselective sulfation at position 3 of the outer galactose residue through the stannylene procedure, led then to 4-methoxybenzyl O-(3-sulfo-beta-D- galactopyranosyl)-(1-->4)-O-(2-acetamido-2-deoxy-6-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl)-(1-->4)-O-beta-D- glucopyranoside disodium salt, which was finally fucosylated using human milk alpha-(1-->3/4)-fucosyltransferase affording, after anomeric deprotection, the target pentasaccharide.     

A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.     

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.     

Identification of a RAPD marker linked to progressive rod-cone degeneration in dogs

1. Tammar Wallaby embryonic blood has been shown to have three alpha-like and two beta-like globin chains in its four haemoglobin components and partial sequences of several chains have been determined. 2. The major embryonic beta-like chain (epsilon) is similar to other mammalian embryonic beta-like chains on the basis of sequencing its first 60 amino acids. 3. There is another embryonic beta-like chain present in one haemoglobin component. It was designated omega and, in its first 54 amino acids, it has features that are more like avian globins than mammalian globins. 4. The one alpha-like embryonic globin sequenced has mammalian rather than avian characteristics. 5. A provisional phylogenetic tree of beta-like globins has been determined. The Tammar epsilon-globin forms a monophyletic group with marsupial and other mammalian embryonic globins; the omega-globin forms a monophyletic group with bird adult and embryonic globins.     

Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.     

Kinetics and mechanism of the cleavage of the peptide bond next to asparagine

The spontaneous cleavage reaction of the tetra-peptide Piv-Gly Asn-Sar-Gly-NHtBu to the C-terminal dipeptide and N-terminal succinimide dipeptide proceeds through pre-equilibrium deprotonation of the amide group of the asparagine side chain, followed by intramolecular nucleophilic attack of nitrogen on the peptide carbonyl carbonyl carbon atom. General acid-catalyzed breakdown of the intermediate then gives the products. According to this mechanism, the reaction rate strongly increases with pH and buffer concentration.     

Telomere length regulation in mice is linked to a novel chromosome locus

Little is known about the mechanisms that regulate species-specific telomere length, particularly in mammalian species. The genetic regulation of telomere length was therefore investigated by using two inter-fertile species of mice, which differ in their telomere length. Mus musculus (telomere length >25 kb) and Mus spretus (telomere length 5-15 kb) were used to generate F1 crosses and reciprocal backcrosses, which were then analyzed for regulation of telomere length. This analysis indicated that a dominant and trans-acting mechanism exists capable of extensive elongation of telomeres in somatic cells after fusion of parental germline cells with discrepant telomere lengths. A genome wide screen of interspecific crosses, using M. spretus as the recurrent parent, identified a 5-centimorgan region on distal chromosome 2 that predominantly controls the observed species-specific telomere length regulation. This locus is distinct from candidate genes encoding known telomere-binding proteins or telomerase components. These results demonstrate that an unidentified gene(s) mapped to distal chromosome 2 regulates telomere length in the mouse.     

Kinetic mechanism of Escherichia coli asparagine synthetase B

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartate, glutamine, and ATP. A combination of kinetic, isotopic-labeling, and stoichiometry studies have been performed to define the nature of nitrogen transfer mediated by AS-B. The results of initial rate studies were consistent with initial binding and hydrolysis of glutamine to glutamate plus enzyme-bound ammonia. The initial velocity results were equally consistent with initial binding of ATP and aspartate prior to glutamine binding. However, product inhibition studies were only consistent with the latter pathway. Moreover, isotope-trapping studies confirmed that the enzyme-ATP-aspartate complex was kinetically competent. Studies using 18O-labeled aspartate were consistent with formation of a beta-aspartyl-AMP intermediate, and stoichiometry studies revealed that 1 equiv of this intermediate formed on the enzyme in the absence of a nitrogen source. Taken together, our results are most consistent with initial formation of beta -aspartyl-AMP intermediate prior to glutamine binding. This sequence leaves open many possibilities for the chemical mechanism of nitrogen transfer.     

Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100-insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.     

Proton transfer reactions linked to rhodopsin activation

Purified bovine rhodopsin solubilized in dodecyl maltoside was photolyzed at 20 degreesC with 477 nm light, and difference spectra were collected at time delays ranging from 10 micros to 10 ms after photolysis. Bromocresol purple was added to the samples to detect pH changes in the aqueous environment due to changes in the protonation state of rhodopsin. The data were analyzed using singular value decomposition and global exponential fitting, which revealed three exponential processes indicating the presence of at least four intermediates. Spectral changes of the indicator dye were separated from those of rhodopsin, and proton release and uptake rates were analyzed within the framework of rhodopsin photoreaction kinetics. Proton release occurred during Lumi decay to Meta-I380 followed by uptake upon Meta-I380 decay and by a more significant proton uptake with the time course of Meta-I480 decay. On the basis of the estimated number of protons released and taken up in each step of the rhodopsin photoreaction, we concluded that two forms of Meta-II are present. The two forms of Meta-II, Meta-IIa' and Meta-IIb, differ in protonation state from one another as do both from the earlier, 380 nm absorbing form, Meta-I380.     

Anaerobic benzene biodegradation linked to nitrate reduction

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [14C]benzene, approximately 92 to 95% of the label was recovered in 14CO2, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.     

CTL epitope generation is tightly linked to cellular proteolysis of a Listeria monocytogenes antigen

Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.