A concise synthesis of two isomeric pentasaccharides, the O repeat units from the lipopolysaccharides of P. syringae pv. porri NCPPB 3364T and NCPPB 3365

A concise synthesis of two isomeric pentasaccharides, alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap (A) and alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap (B), the O repeats from the lipopolysaccharides of Pseudonomonas syringae pv. porri NCPPB 3364T and 3365 was achieved via assembly of the building blocks, allyl 3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (1), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2), allyl 4-O-benzoyl-3-O-chloroacetyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (10). Coupling of 1 with 2 followed by deallylation and trichloroacetimidate formation gave the disaccharide donor 5, while condensation of 6 with 7, followed by dechloroacetylation, offered the disaccharide acceptor 9. Then, 5 was coupled with 10 to obtain the trisaccharide 11, and subsequent deallylation and trichloroacetimidate formation furnished the trisaccharide donor 13. Coupling of 9 with 13, followed by deprotection, afforded pentasaccharide 19, while condensation of 9 with 5, followed by deallylation and trichloroacetimidate formation, gave the tetrasaccharide donor 16, whose coupling with 10 and subsequent deprotection yielded another pentasaccharide 22.     

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.     

Synthesis of an xylosylated rhamnose pentasaccharide, the repeating unit of the O-chain polysaccharide of the lipopolysaccharide of Xanthomonas campestris pv. begoniae GSPB 525

A xylosylated rhamnose pentasaccharide, alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->2)-]-alpha-L-Rhap-(1-->3)-[beta-L-Xylp-(1-->4)]-L-Rhap, the repeating unit of the O-chain polysaccharide (OPS) of the lipopolysaccharides of Xanthomonas campestris pv. begoniae GSPB 525 was synthesized by a highly regio- and stereoselective way. Thus coupling of 1,2-O-ethylidene-beta-L-rhamnopyranose (1) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2) to give (1-->3)-linked disaccharide (3), subsequent benzoylation, deethylidenation, acetylation, 1-O-deacetylation, and trichloroacetimidation afforded the disaccharide donor 11. Condensation of 11 with 1 yielded 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2-O-acetyl-4-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-1,2-O-ethylidene-beta-L-rhamnopyranose (12), and selective deacetylation of 12 yielded the trisaccharide diol acceptor 15. Coupling of 15 with 2,3,4-tri-O-benzoyl-alpha-L-xylopyranosyl trichloroacetimidate (16), followed by deprotection, gave the target pentasaccharide 19.     

Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.     

Flagellin glycans from two pathovars of Pseudomonas syringae contain rhamnose in D and L configurations in different ratios and modified 4-amino-4,6-dideoxyglucose

Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and (1)H and (13)C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Synthesis of heparin partial structures and their binding activities to platelets

A synthetic pentasaccharide corresponding to the antithrombin III-binding region in heparin was also found to bind to human platelets. To identify the platelet-binding site in the pentasaccharide which is expected to be a novel sequence in heparin responsible for its platelet-binding, five partial structures of this particular pentasaccharide were synthesized. In a competitive assay using [3H]-heparin, a trisaccharide, O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-glucopyranosyl)-1--> 4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1-->4)-2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranose, was concluded to be a high-affinity site for heparin's binding to platelets.     

The structure of the O-specific polysaccharide of Escherichia coli O117:K98:H4

The primary structure of the O-antigen of Escherichia coli O117 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear pentasaccharide repeating units with the structure: -->3)-alpha-D-GalpNAc-(1-->4)-beta-D-GalpNAc-(1-->3)-alpha-L-Rhap- (1-->4)- alpha-D-Glcp-(1-->4)-beta-D-Galp-(1-->     

Structure of the O-specific polysaccharide of Mesorhizobium huakuii IFO15243T

The structure of the O-specific polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Mesorhizobium huakuii IFO15243T was studied using methylation analysis and various one- and two-dimensional 1H and 13C NMR experiments. The O-antigen polysaccharide was found to be linear polymer constituted by a trisaccharide repeating unit of the following structure: --> 2)-alpha-L-6dTalp-(1 --> 3)-alpha-L-6dTalp-(1 --> 2)-alpha-L-Rhap-(1 -->.     

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.     

A facile regio- and stereoselective synthesis of mannose octasaccharide of the N-glycan in human CD2 and mannose hexasaccharide antigenic factor 13b

A highly concise and effective synthesis of the mannose octasaccharide of the N-linked glycan in the adhesion domain of human CD2 was achieved via TMSOTf-promoted selective 6-glycosylation of a trisaccharide 4,6-diol acceptor with a pentasaccharide donor, followed by deprotection. The pentasaccharide was constructed by selective 3,6-diglycosylation of 1,2-O-ethylidene-beta-D-mannopyranose with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, while the trisaccharide was obtained by selective 3-O-glycosylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside with the same disaccharide trichloroacetimidate, followed by debenzylidenation. The mannose hexasaccharide antigenic factor 13b was synthesized by condensation of a trisaccharide donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate, with a trisaccharide acceptor, methyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside, followed by deprotection.     

Contribution of hydrogen bonding to the conformational stability of ribonuclease T1.

For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1 (RNase T1). When RNase T1 folds, 86 intramolecular hydrogen bonds with an average length of 2.95 A are formed. Twelve mutants of RNase T1 [Tyr----Phe (5), Ser----Ala (3), and Asn----Ala (4)] have been prepared that remove 17 of the hydrogen bonds with an average length of 2.93 A. On the basis of urea and thermal unfolding studies of these mutants, the average decrease in conformational stability due to hydrogen bonding is 1.3 kcal/mol per hydrogen bond. This estimate is in good agreement with results from several related systems. Thus, we estimate that hydrogen bonding contributes about 110 kcal/mol to the conformational stability of RNase T1 and that this is comparable to the contribution of the hydrophobic effect. Accepting the idea that intramolecular hydrogen bonds contribute 1.3 +/- 0.6 kcal/mol to the stability of systems in an aqueous environment makes it easier to understand the stability of the "molten globule" states of proteins, and the alpha-helical conformations of small peptides.     

Synthesis of an alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6

A hexasaccharide, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->2)]-D-Glcp, the alpha-linked dimer of the trisaccharide repeating unit of the exopolysaccharide produced by Pediococcus damnosus 2.6, was synthesized as its methyl glycoside. Condensation of fully benzoylated alpha-D-glucopyranosyl trichloroacetimidate (1) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) selectively furnished (1-->3)-linked disaccharide 3, and subsequent 2-O-acetylation, desulfation, and trichloroacetimidate formation afforded the disaccharide donor 6. Meanwhile, selective 3-O-coupling of methyl 4,6-O-benzylidene-alpha-d-glucopyranoside (8) with 3-O-allyl-2,4,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (7), followed by coupling with 1 gave the trisaccharide 10. Removal of the benzylidene group of 10, benzoylation, and deallylation produced the trisaccharide acceptor 12. Condensation of 12 with 6 yielded a pentasaccharide mixture 13 with beta and alpha isomers in a ratio of 2:1. Removal of the benzylidene group of 13, followed by benzoylation gave the pentasaccharide mixture 14. Selective 2'-deacetylation of the isolated beta-linked 14beta with MeCOCl/MeOH/CH2Cl2 did not give the expected pentasaccharide acceptor, and serious decomposition occurred, indicating a large steric hindrance at C-2'. Alternatively, 2,3-di-O-glycosylation of allyl 4,6-O-benzylidene-beta-D-glucopyranoside (21) with 1 gave 22, then deallylation and trichloroacetimidate formation afforded the trisaccharide donor 24. Condensation of 12 with 24 furnished only the alpha-linked hexasaccharide 25, and its deprotection gave the free hexaoside 27.     

Antibody variable region binding by Staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (V(H)), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3-4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5+/-0.5 x 10(5) M(-1). A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0+/-0.1, Ka = 2.0+/-0.3 x 10(5) M(-1), and deltaH = -7.1+/-0.4 kcal mol(-1). Similar binding thermodynamics are obtained for titration of the isolated V(H) domain with E-domain indicating that the E-domain binding site on Fab resides within V(H). E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2+/-0.1, Ka > 1.0 x 10(7) M(-1), and deltaH = -24.6+/-0.6 kcal mol(-1). Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the V(H) domain implicated previously in protein A binding.     

Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes.

Values of K, delta G(o), delta H(o), delta S(o) and delta C(po) for the binding reaction of small organic ligands forming 1:1 complexes with either alpha- or beta-cyclodextrin were obtained by titration calorimetry from 15 degrees C to 45 degrees C. A hydrogen bond or hydrophobic interaction was introduced by adding a single functional group to the ligand. The thermodynamics of binding with and without the added group are compared to estimate the contribution of the hydrogen bond or hydrophobic interaction. A change in the environment of a functional group is required to influence the binding thermodynamics, but molecular size-dependent solute-solvent interactions have no effect. For phenolic O-H-O hydrogen bond formation, delta H(o) varies from -2 to -1.4 kcal mol(-1) from 15 degrees C to 45 degrees C, and delta C(p) is increased by 18 cal K(-1) mol(-1). The hydrophobic interaction has an opposite effect: in alpha-cyclodextrin, delta C(po) = -13.3 cal K(-1) mol(-1) per ligand -CH(2)-, identical to values found for the transfer of a -CH(2)-group from water to a nonpolar environment. At room temperature, the hydrogen bond and the -CH(2)-interaction each contribute about -600 cal mol(-1) to the stability (delta G(o)) of the complex. With increased temperature, the hydrogen bond stability decreases (i.e., hydrogen bonds "melt"), but the stability of the hydrophobic interaction remains essentially constant.     

Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus

The seed lectin (DBL) from the leguminous plant Dolichos biflorus has a unique specificity among the members of the legume lectin family because of its high preference for GalNAc over Gal. In addition, precipitation of blood group A+H substance by DBL is slightly better inhibited by a blood group A trisaccharide (GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal) containing pentasaccharide, and about 40 times better by the Forssman disaccharide (GalNAc(alpha1-3)GalNAc) than by GalNAc. We report the crystal structures of the DBL-blood group A trisaccharide complex and the DBL-Forssman disaccharide complex.A comparison with the binding sites of Gal-binding legume lectins indicates that the low affinity of DBL for Gal is due to the substitution of a conserved aromatic residue by an aliphatic residue (Leu127). Binding studies with a Leu127Phe mutant corroborate these conclusions. DBL has a higher affinity for GalNAc because the N-acetyl group compensates for the loss of aromatic stacking in DBL by making a hydrogen bond with the backbone amide group of Gly103 and a hydrophobic contact with the side-chains of Trp132 and Tyr104.Some legume lectins possess a hydrophobic binding site that binds adenine and adenine-derived plant hormones, i.e. cytokinins. The exact function of this binding site is unknown, but adenine/cytokinin-binding legume lectins might be involved in storage of plant hormones or plant growth regulation. The structures of DBL in complex with adenine and of the dimeric stem and leaf lectin (DB58) from the same plant provide the first structural data on these binding sites. Both oligomers possess an unusual architecture, featuring an alpha-helix sandwiched between two monomers. In both oligomers, this alpha-helix is directly involved in the formation of the hydrophobic binding site. DB58 adopts a novel quaternary structure, related to the quaternary structure of the DBL heterotetramer, and brings the number of know legume lectin dimer types to four.     

Forces stabilizing proteins

The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH₂− group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.     

N-acetylmuramic acid as capping element of alpha-D-fucose-containing S-layer glycoprotein glycans from Geobacillus tepidamans GS5-97T

Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Fucp-(1-->](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core -->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1--> to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).     

Free-energy simulations reveal that both hydrophobic and polar interactions are important for influenza hemagglutinin antibody binding

Antibodies binding to conserved epitopes can provide a broad range of neutralization to existing influenza subtypes and may also prevent the propagation of potential pandemic viruses by fighting against emerging strands. Here we propose a computational framework to study structural binding patterns and detailed molecular mechanisms of viral surface glycoprotein hemagglutinin (HA) binding with a broad spectrum of neutralizing monoclonal antibody fragments (Fab). We used rigorous free-energy perturbation (FEP) methods to calculate the antigen-antibody binding affinities, with an aggregate underlying molecular-dynamics simulation time of several microseconds (～2 μs) using all-atom, explicit-solvent models. We achieved a high accuracy in the validation of our FEP protocol against a series of known binding affinities for this complex system, with <0.5 kcal/mol errors on average. We then introduced what to our knowledge are novel mutations into the interfacial region to further study the binding mechanism. We found that the stacking interaction between Trp-21 in HA2 and Phe-55 in the CDR-H2 of Fab is crucial to the antibody-antigen association. A single mutation of either W21A or F55A can cause a binding affinity decrease of ΔΔG > 4.0 kcal/mol (equivalent to an ～1000-fold increase in the dissociation constant K_d). Moreover, for group 1 HA subtypes (which include both the H1N1 swine flu and the H5N1 bird flu), the relative binding affinities change only slightly (< ±1 kcal/mol) when nonpolar residues at the αA helix of HA mutate to conservative amino acids of similar size, which explains the broad neutralization capability of antibodies such as F10 and CR6261. Finally, we found that the hydrogen-bonding network between His-38 (in HA1) and Ser-30/Gln-64 (in Fab) is important for preserving the strong binding of Fab against group 1 HAs, whereas the lack of such hydrogen bonds with Asn-38 in most group 2 HAs may be responsible for the escape of antibody neutralization. These large-scale simulations may provide new insight into the antigen-antibody binding mechanism at the atomic level, which could be essential for designing more-effective vaccines for influenza.     

Glucuronic acid- and branched sugar-containing glycolipid antigens of Mycobacterium avium

The pentasaccharide hapten of the dominant glycopeptidolipid antigen of serovariant 19 of the Mycobacterium avium complex is noteworthy because of the uniqueness of its distal glycobiose, the presumed antigen determinant, which contains a 3,4-di-O-methyl glucuronic acid and a novel branched sugar. The detailed structure of the entire pentasaccharide has been established by high field 1H and 13C NMR, fast atom bombardment/mass spectrometry, and various specific degradations as 3,4-di-O-Me-beta-D-GlcAp-(1----3)-2,4-di-O-Me-3-C-Me-3,6-dideox yhexosyl-(1----3)-alpha-L-Rhap-(1----3)-alpha-L-Rhap-( 1----2)-6-dT al; the extreme acid lability of the novel penultimate sugar presented special structural challenges. Thus, the task of defining the variable epitopes of M. avium serovariants in order to charter the epidemiology of opportunistic mycobacterial diseases continues to reveal an unexpected order of sugar diversity and complexity.