Biomedical subjects
Publications and source records attributed to A Tramontano.
To assess how automatic function assignment will contribute to genome annotation in the next five years, we have performed an analysis of 31 available genome sequences. An emerging pattern is that function can be predicted for almost two-thirds of the 73,500 genes that were analyzed. Despite progress in computational biology, there will always be a great need for large-scale experimental determination of protein function.
The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.
Several experimental studies on hepatitis C virus (HCV) have suggested the envelope glycoprotein E2 as a key antigen for an effective vaccine against the virus. Knowledge of its structure, therefore, would present a significant step forward in the fight against this disease. This paper reports the application of fold recognition methods in order to produce a model of the HCV E2 protein. Such investigation highlighted the envelope protein E of Tick Borne Encephalitis virus as a possible template for building a model of HCV E2. Mapping of experimental data onto the model allowed the prediction of a composite interaction site between E2 and its proposed cellular receptor CD81, as well as a heparin binding domain. In addition, experimental evidence is provided to show that CD81 recognition by E2 is isolate or strain specific and possibly mediated by the second hypervariable region (HVR2) of E2. Finally, the studies have also allowed a rough model for the quaternary structure of the envelope glycoproteins E1 and E2 complex to be proposed. Proteins 2000;40:355-366.
Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.
We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.
A spleen cDNA library was constructed from the Antarctic teleost Trematomus bernacchii and immunoscreened with rabbit IgG specific for T. bernacchii Ig heavy chain. Eleven cDNA clones, varying in size and encoding the entire heavy chain or parts of it, were isolated. Here the complete nucleotide and deduced amino acid sequences of clone 2C2 encoding the secretory IgH chain form are reported. Comparison of the amino acid sequence of the entire constant region of the T. bernacchii Ig heavy chain with those from other teleosts and two holostean fish showed percent identity ranging 53.6-60.6%, with the highest values found for Salmoniformes. The multiple sequence alignment revealed the presence of two remarkable insertions: one at the VH-CH1 boundary and a second one, not found in any other IgM heavy chain, localised at the CH2-CH3 boundary. The latter occurred in the region proposed to act as a 'hinge', and resulted in a CH2-CH3 hinge peptide longer than any other IgM hinge. Differences were also found in the number and position of putative N-glycosylation sites of the compared sequences. It is suggested that the unusual features found in the T. bernacchii Ig heavy chain might contribute to the flexibility of the Ig molecule and help understand more about the adaptation of Ig molecules to the polar sea environment.
Our understanding of the rules relating sequence to structure in antibodies has led to the development of accurate knowledge-based procedures for antibody modeling. Information gained from the analysis of antibody structures has been successfully exploited to engineer antibody-like molecules endowed with prescribed properties, such as increased stability or different specificity, many of which have a broad spectrum of applications both in therapy and in research. Here we describe a knowledge-based procedure for the prediction of the antibody-variable domains, based on the canonical structures method for the antigen-binding site, and discuss its expected accuracy and limitations. The rational design of antibody-based molecules is illustrated using as an example one of the most widely employed modifications of antibody structures: the humanization of animal-derived antibodies to reduce their immunogenicity for serotherapy in humans.
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Identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. One route to this end is the generation and screening of random libraries of cleavage sites. Both synthetic and phage-displayed libraries have been extensively used in vitro. We describe a novel system based on recombinant Sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzyme and overcoming the problem of choosing the correct in vitro conditions. As a model we used the serine protease of the hepatitis C virus (HCV). We engineered the gene coding for this enzyme and two specific cleavage sites in the Sindbis virus structural gene and constructed libraries of viral genomes with a random sequence at either of the cleavage sites. The system was designed so that only viral genomes coding for sequences cleaved by the protease would produce viable viruses. With this system we selected viruses containing sequences mirroring those of the natural HCV protease substrates which were cleaved with comparable efficiencies.
The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.
Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.
This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.
The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.
When a protein sequence does not share any significant sequence similarity with a protein of known structure, homology modeling cannot be applied. However, many novel and interesting methods, such as secondary structure prediction, fold recognition, and prediction of long-range interactions, are being developed and have been shown to be reasonably successful in predicting protein structures from sequence data and evolutionary information. The a priori evaluation of the correctness of a prediction obtained by one of these methods is however often problematic. Consequently, it is important to use all available information provided by as many different methods as possible and all the available experimental data about the protein of interest, since the consistency of the results is indicative of the reliability of the prediction. Hence the need has arisen for suitable tools able to compare results provided by different methods and evaluate their consistency. We have therefore constructed GLASS, a general platform to read, visualize, compare, and evaluate prediction results from many different sources and to project these prediction results into three dimensions. In addition, GLASS allows the comparison of selected parameters calculated for a model with the distribution observed in real protein structures, thus providing an easy way to test new methods for evaluating the likelihood of different structural models. GLASS can be considered as a "workbench" for structural predictions useful to both experimentalists and theoreticians.
Antigen-combining sites of antibodies are constructed from six loops from VL and VH domains. The third hypervariable region of the heavy chain is far more variable than the others in length, sequence and structure, and was not included in the canonical-structure description of the conformational repertoire of the three hypervariable regions of V kappa chains and the first two of VH chains. Here we present an analysis of the conformations of the third hypervariable region of VH domains (the H3 regions) in antibodies of known structure. We define the H3 region as comprising the residues between 92Cys and 104Gly. We divide it into a torso comprising residues proximal to the framework, four residues from the N terminus and six residues from the C terminus, and a head. There are two major classes of H3 structures that have more than ten residues between 92Cys and 104Gly: (1) the conformation of the torso has a beta-bulge at residue 101, and (2) the torso does not contain a bulge, but continues the regular hydrogen-bonding pattern of the beta-sheet hairpin. The choice of bulged versus non-bulged torso conformation is dictated primarily by the sequence, through the formation of a salt bridge between the side-chains of an Arg or Lys at position 94 and an Asp at position 101. Thus the torso region appears to have a limited repertoire of conformations, as in the canonical structure model of other antigen-binding loops. The heads or apices of the loops have a very wide variety of conformations. In shorter H3 regions, and in those containing the non-bulged torso conformation, the heads follow the rules relating sequence to structure in short hairpins. We surveyed the heads of longer H3 regions, finding that those with bulged torsos present many very different conformations of the head. We recognize that H3, unlike the other five antigen-binding loops, has a conformation that depends strongly on the environment, and we have analysed the interactions of H3 with residues elsewhere in the VH domain, in the VL domain, and with ligands, and their effects on the conformation of H3. We tested these results by attempts to predict the conformations of H3 regions in antibody structures solved after the results were derived. The general conclusion of this work is that the conformation of H3 shows some regularities, from which rules relating sequence to conformation can be stated, but to a less complete degree than for the other five antigen-binding loops. Accurate prediction of the torso conformation is possible in most cases; predictions of the conformation of the head is possible in some cases. However, our understanding of the sequence-structure relationships has reduced the uncertainty to no more than a few residues at the apex of the H3 region.
The field of protein structure prediction is evolving rapidly and in the last few years a number of new methods have been developed and evaluated. However, comparative modeling, or modeling by homology, is still the method of choice when the unknown protein shares any significant sequence similarity with a protein of known structure. The accuracy of the method is highly dependent on the degree of similarity between the target protein and that used as a template. Nevertheless, careful consideration of all the steps performed in the modeling procedure allows useful information to be obtained also from a model based on very low sequence identity.
A monoclonal antibody (MAb) was produced against the p-nitrophenylphosphate derivative of 3 alpha, 5 beta-lithocholic acid, a transition-state analog for hydrolysis of a steroidal p-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants kcat = 4.0 x 10(-2)/min and K(m) = 3.3 microM at pH 9.0 and 35 degrees C. The Ab also hydrolyzed the isomeric p-nitrophenylcarbonate of 3 beta, 5 beta-lithocholic acid with kcat = 8.4 x 10(-2)/min and K(m) = 1.0 microM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 microM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC50 ca. 40 microM), the Ab-catalyzed reaction was completely inhibited at less than 1 microM of the TSA. The relative rates and efficiencies of the MAb-catalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.
BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.