Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.     

High-speed running performance is largely unaffected by hypoxic reductions in aerobic power.

We tested the importance of aerobic metabolism to human running speed directly by altering inspired oxygen concentrations and comparing the maximal speeds attained at different rates of oxygen uptake. Under both normoxic (20.93% O2) and hypoxic (13.00% O2) conditions, four fit adult men completed 15 all-out sprints lasting from 15 to 180 s as well as progressive, discontinuous treadmill tests to determine maximal oxygen uptake and the metabolic cost of steady-state running. Maximal aerobic power was lower by 30% (1.00 +/- 0.15 vs. 0.77 +/- 0.12 ml O2. kg-1. s-1) and sprinting rates of oxygen uptake by 12-25% under hypoxic vs. normoxic conditions while the metabolic cost of submaximal running was the same. Despite reductions in the aerobic energy available for sprinting under hypoxic conditions, our subjects were able to run just as fast for sprints of up to 60 s and nearly as fast for sprints of up to 120 s. This was possible because rates of anaerobic energy release, estimated from oxygen deficits, increased by as much as 18%, and thus compensated for the reductions in aerobic power. We conclude that maximal metabolic power outputs during sprinting are not limited by rates of anaerobic metabolism and that human speed is largely independent of aerobic power during all-out runs of 60 s or less.     

Identification of the A and M antigens of Brucella as the O-polysaccharides of smooth lipopolysaccharides

Rabbit antisera, cross-absorbed serotype-specific for the Brucella A and M antigens, precipitated respectively the smooth lipopolysaccharides from B. abortus 1119-3 and B. melitensis 16M. The antigenic A and M activity of these lipopolysaccharides was shown to reside within the O-chain region of the smooth lipopolysaccharides by inhibition experiments. Homologous O-polysaccharides showed the highest inhibitory activity in ELISA, although both the A and M antigens were active as heterologous inhibitors, showing that the antigenic determinants of the classical A and M antigens are therefore within the respective O-polysaccharide structures. Their cross-serological activity may be explained in terms of the distinct but related chemical structures of these polysaccharides.     

Antibody to an artificial disaccharide antigen cross-reactive with Neisseria gonorrhoeae lipopolysaccharide.

An artificial antigen was prepared from 4-O-beta-I-galactopyranosyl-D-glucose (lactose) and 8-ethoxycarbonyloctanol. Covalent attachment to bovine serum albumin provided an antigen that elicited antilactose antibody in rabbits and goat. These antibodies were active against Neisseria gonorrhoeae lipopolysaccharide in passive hemagglutination tests. The same antibody agglutinated cells of Streptococcus faecalis, strain N, and precipitated the lactose-containing cell wall diheteroglycan of this organism. Fractionation of rabbit and goat antibody raised against the synthetic antigen of S. faecalis vaccine provided two antibody fractions only one of which, eluted from the immunoadsorbent by galactose, was active against N. gonorrhoeae lipopolysaccharide.     

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.     

A metabolic basis for impaired muscle force production and neuromuscular compensation during sprint cycling

For both different individuals and modes of locomotion, the external forces determining all-out sprinting performances fall predictably with effort duration from the burst maximums attained for 3 s to those that can be supported aerobically as trial durations extend to roughly 300 s. The common time course of this relationship suggests a metabolic basis for the decrements in the force applied to the environment. However, the mechanical and neuromuscular responses to impaired force production (i.e., muscle fatigue) are generally considered in relation to fractions of the maximum force available, or the maximum voluntary contraction (MVC). We hypothesized that these duration-dependent decrements in external force application result from a reliance on anaerobic metabolism for force production rather than the absolute force produced. We tested this idea by examining neuromuscular activity during two modes of sprint cycling with similar external force requirements but differing aerobic and anaerobic contributions to force production: one- and two-legged cycling. In agreement with previous studies, we found greater peak per leg aerobic metabolic rates [59% (+/-6 SD)] and pedal forces at VO2 peak [30% (+/-9)] during one- vs. two-legged cycling. We also determined downstroke pedal forces and neuromuscular activity by surface electromyography during 15 to 19 all-out constant load sprints lasting from 12 to 400 s for both modes of cycling. In support of our hypothesis, we found that the greater reliance on anaerobic metabolism for force production induced compensatory muscle recruitment at lower pedal forces during two- vs. one-legged sprint cycling. We conclude that impaired muscle force production and compensatory neuromuscular activity during sprinting are triggered by a reliance on anaerobic metabolism for force production.     

Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.

The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.     

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.     

Very different performance of the power Doppler modalities of several ultrasound machines ascertained by a microvessel flow phantom

Introduction

In many patients with rheumatoid arthritis (RA) subclinical disease activity can be detected with ultrasound (US), especially using power Doppler US (PDUS). However, PDUS may be highly dependent on the type of machine. This could create problems both in clinical trials and in daily clinical practice. To clarify how the PDUS signal differs between machines we created a microvessel flow phantom.

Methods

The flow phantom contained three microvessels (150, 1000, 2000 microns). A syringe pump was used to generate flows. Five US machines were used. Settings were optimised to assess the lowest detectable flow for each US machine.

Results

The minimal detectable flow velocities showed very large differences between the machines. Only two of the machines may be able to detect the very low flows in the capillaries of inflamed joints. There was no clear relation with price. One of the lower-end machines actually performed best in all three vessel sizes.

Conclusions

We created a flow phantom to test the sensitivity of US machines to very low flows in small vessels. The sensitivity of the power Doppler modalities of 5 different machines was very different. The differences found between the machines are probably caused by fundamental differences in processing of the PD signal or internal settings inaccessible to users. Machines considered for PDUS assessment of RA patients should be tested using a flow phantom similar to ours. Within studies, only a single machine type should be used.