Biomedical subjects
Publications and source records attributed to A R Rees.
The antigen-binding fragments of four monoclonal antibodies that cross-react with both the "loop" peptide of hen egg-white lysozyme (residues 57 to 84) against which they were raised, and with the native protein (HEL) have been crystallized. One of these fragments also crystallizes as a complex with the peptide antigen.
A pre-steady-state multiple-turnover kinetic burst is observed during hydrolysis of phenyl acetate by the catalytic antibody, 20G9. The burst is caused by partial product inhibition by phenol (Ki,app = 2.5 microM), which lowers both kcat and KM by almost an order of magnitude without affecting kcat/KM. The acid limb of the steady-state kcat pH profile of native 20G9 has a pKa of 9.6, suggesting a catalytic role for tyrosine. Additional evidence for an essential tyrosyl residue is that mild treatment of 20G9 with tetranitromethane nitrates a single tyrosine per equivalent of antigen binding sites and the mononitrated derivative has less than 5% of the native activity. Near-UV absorbance spectroscopy suggests that the alternative substrates N-carbobenzoxyglycine O-phenyl ester (ZG-OPh) and N-acetylglycine O-phenyl ester (AcG-OPh) acylate multiple tyrosines on the antibody. Neither ZG-OPh nor AcG-OPh are measurably catalyzed once appreciable acylation has taken place. Antibody acylated by ZG-OPh is inactive toward phenyl acetate hydrolysis, but can be reactivated by hydroxylamine. The data and derived kinetic rate equations are consistent with an acyl mechanism for phenyl acetate hydrolysis in which phenol inhibits by binding to a covalent O-acetyltyrosyl intermediate, slowing deacylation. Although the data are consistent with such a mechanism, they do not rule out other plausible, yet less unifying mechanisms of phenol inhibition; the observed burst could conceivably result from partial mixed phenol inhibition or from phenol-induced nonproductive substrate binding. Because antibodies often use tyrosines in antigen binding, tyrosyl catalytic antibodies may be commonly encountered in the future.
The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.
The time course of phenylacetate hydrolysis by the catalytic antibody 20G9 has a kinetic burst lasting several reaction cycles. The burst is caused by partial mixed inhibition by one product of the hydrolysis, phenol, which binds with an apparent dissociation constant of 4.6 microM. Phenol binding causes kcat to decrease from 9.1 min-1 to 1.0 min-1 and Km to decrease from 300 microM to 36 microM. Because Km decreases but kcat/Km is unaffected, phenol must perturb the ground state structure but not the transition state structure. Structural complementarity to the transition state seems to be an important contributor to catalysis by 20G9 because weak binding in the ground state can be markedly improved by adding phenol, but tight binding of the transition state, which has been optimized by the immune system, cannot be readily improved. Further evidence that the substrate ground and transition states differ greatly in complementarity to the antibody is that the substrate binds more than five orders of magnitude more weakly than the transition state analogue hapten to which the antibody was raised. Two additional phenol molecules bind at higher product concentrations; the first binds over the concentration range of 15 to 86 microM and accelerates hydrolytic activity by 42%; the second is a competitive inhibitor with a Ki of 140 microM. Binding of multiple phenol molecules suggests the presence of abundant hydrophobic amino acids in the complementarity-determining region of 20G9.
Each of the six CDRs of Gloop2 is shown with the modeled structure in. Overall, the results obtained using the combined algorithm are similar in accuracy to those achieved using the canonical method of Chothia et al. However, the canonical method is limited to those loops where the key residues identified by Chothia are present. With the number of antibody structures currently available, it is not possible to classify CDR-H3 into canonical ensembles. Additionally, a small percentage of examples in the remaining CDRs do not match the current canonical classifications and the protein engineer may well wish to mutate the key residues, precluding the use of Chothia's method for modeling the resulting conformation. Thus the best approach appears to be to use Chothia's method (at least to model the backbone conformation) when the loop to be modeled is represented in the database of canonical structures. Any other loops, either unrepresented among the known canonicals (including CDR-H3), or where mutations have been made to the key residues, may then be modeled by the combined algorithm presented here.
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Catalytic antibodies are introduced as an important new class of biomolecules for molecular recognition in biosensors in which the binding sites are continually regenerated by the catalytic reaction of the substrate. Consequently, molecular recognition by catalytic antibodies can yield reversible immunoblosensors. In this example, a prototype potentiometric biosensor is described in which a micro-pH electrode is modified with a catalytic antibody that catalyzes the hydrolysis of phenyl acetate, producing hydrogen ions that can be monitored by the electrode. The reversible response is linear with the log of substrate concentration over a range of 20-500 microM with a detection limit of 5 microM under the conditions of this study. Alternative applications of catalytic antibodies in other biosensor configurations are discussed.
The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.
Kawasaki disease is a systemic vasculitis in which secondary development of coronary artery aneurysms can occur. Because Factor VIII related antigen has been found increased in other vasculiditides, VIII R:Ag was measured serially in patients with Kawasaki disease. Factor VIII related antigen was prospectively evaluated in the acute phase of ten patients with Kawasaki disease, all of whom showed increased values at this stage (p greater than 0001). In six children a second sample was drawn at the convalescent phase, and all were normal. Of the original ten patients, two developed coronary artery aneurysms. Acute Factor VIII related antigen levels were not higher nor did Factor VIII related antigen fail to return to baseline in these two patients. Based on our findings, Factor VIII related antigen is elevated in the acute phase of Kawasaki disease and returns to normal levels in the convalescent phase.
For maximum value, a predicted model of an antibody-combining site should have an accuracy approaching that of an X-ray structure (1.6-2.7 A). In addition, the method by which the combining site is modelled should make no demands on the user of a sort that require arbitrary or subjective decisions to be made during the process. We have made substantial progress towards this objective and some recent results are reviewed. In addition, we describe how the modelling protocols developed can aid in the design of novel features within the antibody-combining site. The particular design example reported here suggests an approach for the introduction of metal-binding sites to create metallo-antibodies. This type of modification may be useful in the design of immunobiosensors, the induction of catalytic activity or simply as an alternative to metal chelates in the preparation of antibodies for imaging.
Quiescent fibroblasts derived from human fetal lung can be stimulated to reinitiate DNA synthesis by sequential addition of 3 nM IGF-1 and a low concentration (8 pM) of EGF or by continuous exposure to 10% fetal calf serum or 10 ng/ml PDGF. Bradykinin blocks the IGF-1 and EGF-dependent signals without affecting the response to serum or PDGF. It activates protein kinase C and its anti-mitogenic effect is abolished after this kinase has been down-regulated. Bradykinin has no effect on the binding affinity of the EGF receptor whereas phorbol ester induces its 'transmodulation' to low affinity.
To be of any value, a predicted model of an antibody combining site should have an accuracy approaching that of antibody structures determined by x-ray crystallography (1.6-2.7 A). A number of modeling protocols have been proposed, which fall into two main categories--those that adopt a knowledge-based approach and those that attempt to construct the hypervariable loop regions of the antibody ab initio. Here we present a combined algorithm requiring no arbitrary decisions on the part of the user, which has been successfully applied to the modeling of the individual loops in two systems: the anti-lysozyme antibody HyHel-5, the crystal structure of which is as a complex with lysozyme [Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C. & Davies, D. R. (1987) Proc. Natl. Acad. Sci. USA 84, 8075-8079], and the free antigen binding fragment (Fab) of the anti-lysozyme peptide antibody, Gloop2. This protocol may be used with a high degree of confidence to model single-loop replacements, insertions, deletions, and side-chain replacements. In addition, it may be used in conjunction with other modeling protocols as a method by which to model particular loops whose conformations are predicted poorly by these methods.
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Two families of homeo box-containing genes have been identified in mammals to date, the Antennapedia- and engrailed-like homeo boxes, based on the sequence similarity to those from Drosophila. Here, we report the isolation of a homeo box-containing gene that belongs to a new family of which there are at least three related genes in the mouse genome. The homeo box of this new gene shows remarkable similarity to the Drosophila Msh homeo box that we designate as the prototype for this family. The gene maps to the proximal end of mouse chromosome 5 and does not cosegregate with any known homeo box-containing gene. We designate this locus Hox-7.1. In situ hybridizations to mouse embryos at different stages show a unique pattern of expression, as compared to other homeo box-containing genes described thus far. Hox-7.1 transcripts are detected in 9.5-day-old embryos in the neural crest, developing limb bud, and visceral arches. Later, this gene is expressed in regions of the face that are derived from neural crest and in the interdigital mesenchymal tissues in both the fore- and hindlimbs.
The V region sequences of two anti-DNA (A52, D42) and two anti-RNA (D44, D444) autoantibodies, derived from lupus prone NZB/NZW F1 female mice, were determined by mRNA sequencing. The sequences had the following features: 1) there was no clear sequence relationship between anti-DNA and anti-RNA antibodies; 2) there were no major similarities between any of the L chain sequences and each VL gene segment belonged to a different mouse VK subgroup; 3) the H chains of the two anti-RNA antibodies showed closely related sequences of VH gene segments and very similar third complementarity determining regions (CDR3); 4) the H chains of the two anti-DNA antibodies had VH segments belonging to different VH gene families but had a unique and similar combination of D segments and junctional sequences, suggesting a common recognition element for Ag and/or for idiotypic regulation in the H chain CDR3; and 5) the VH gene segment of one anti-DNA antibody (D42) was found to be very similar to the VH gene segment of a CBA mouse hybridoma antibody (6G6) which binds to the environmental Ag phosphocholine. The three-dimensional structure of the Fv-region of the anti-DNA antibody (D42) was modeled by computer and a stretch of poly(dT), ssDNA was docked to a cleft in the antibody combining site, formed by the three H chain CDR and by CDR1 and CDR3 of the L chain. The cleft is characterized by a preponderance of arginine and tyrosine residues, lining both the walls and base of the cleft.
A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed.
Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.
Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.