Biomedical subjects

D Altschuh

Publications and source records attributed to D Altschuh.

At least 19 recordsLinked to original sources

PubMed2003

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates.

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.

Amino Acid Sequence↗

PubMed2001

BIACORE data processing: an evaluation of the global fitting procedure.

Data from real-time molecular interaction analysis using BIACORE are currently evaluated by numerical integration. We have investigated the ability of two software packages (BIAevaluation 3.0 and CLAMP99) to analyze complex interactions. Three experimental data sets of high quality obtained with BIACORE upgraded and 2000 instruments, representative of simple bimolecular, heterogeneous ligand, and mass-transport-limited interactions, were processed by the global fitting procedure. The two software, which differ mainly in the statistical assessment of the output values, were able to discriminate correctly between various interacting models and provided very close output parameters with satisfactory statistical tests.

Amino Acid Sequence↗

PubMed2001

Delineation of a linear epitope by multiple peptide synthesis and phage display.

Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P, specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composition of a functional epitope. Kinetic measurements of the interaction between peptide variants and the antibody fragment were used to further refine the molecular basis of binding activity. Our results show that the functional epitope of Fab 57P requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. The phage display and Spot synthesis methods allowed the straightforward localization of the binding region and the identification of residues that are essential for recognition. However, these methods yielded slightly different views of accessory factors that are able to influence antibody binding. The influence on binding activity of these factors can only be assessed through quantitative affinity measurements.

Animals↗

PubMed2000

Covariance analysis of protein families: the case of the variable domains of antibodies.

A nonrestrictive method for identifying covariance in protein families is described and applied to human and mouse germline Vkappa and VH sequence alignments. Amino acids that occur at each position in a sequence alignment are divided into two sets, called a word, by generating all possible combinations of alternative amino acids. Each word is associated with a pattern of changes. Words with identical patterns identify covariant positions. In antibody variable domains, the number of words generated ranged between 1103 and 2195 depending on the alignment, of which 4 to 12 % occurred in covariant pairs. Despite the nonrestrictive character of pattern generation, covariant residues did not reflect a random selection with respect to the nature of amino acid changes and/or their spatial proximity in a reference crystallographic structure. This approach allowed the identification of a covariance signal for positions with high variability, mostly located in the outer part of the common structural framework of antibody variable domains. Covariance in these regions may reflect the existence of alternative and mutually exclusive atomic arrangements that are compatible with antibody function. The method may be of general applicability to rationalize residue variability in protein families.

Algorithms↗

PubMed1999

Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein.

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.

Antibody Affinity↗

PubMed1998

Measurement of antigen-antibody interactions with biosensors.

The introduction in 1990 of a new biosensor technology based on surface plasmon resonance has revolutionized the measurement of antigen-antibody binding interactions. In this technique, one of the interacting partners is immobilized on a sensor chip and the binding of the other is followed by the increase in refractive index caused by the mass of bound species. The following immunochemical applications of this new technology will be described: (1) functional mapping of epitopes and paratopes by mutagenesis; (2) analysis of the thermodynamic parameters of the interaction; (3) measurement of the concentration of biologically active molecules; (4) selection of diagnostic probes.

Animals↗

PubMed1997

Cooperative effects of mutations in a recombinant Fab on the kinetics of antigen binding.

Recombinant Fabs, 57P and 174P, recognizing peptide 134-151 of the coat protein of tobacco mosaic virus, differ by 15 amino acid changes in the sequence of their variable region. Kinetic analysis using BIAcore showed that they recognized five peptide variants in the same ranking order, but that Fab 174P consistently dissociated faster from the peptides compared to Fab 57P. In order to identify amino acid substitutions that are responsible for differences in dissociation rates of the two Fabs, six hybrid Fabs have been constructed by exchanging three DNA segments. Four single and five multiple mutants were obtained by site-directed mutagenesis. All Fabs recognized variant peptides in a similar ranking order. The high precision of biosensor measurements made it possible to detect small contributions to dissociation kinetics of at least five substitutions, as well as the presence of small-magnitude non-additive effects of multiple substitutions. Our results demonstrate the cooperative influence on dissociation kinetics of amino acid residues located away from each other and away from the Fab combining site.

Antigens↗

PubMed1996

Functional mapping of conserved residues located at the VL and VH domain interface of a Fab.

The interface between the VL and VH domains of antibodies is highly conserved. To investigate the influence of conserved interface residues on Fab function, 13 interface residues were subjected to codon-based combinatorial alanine scanning mutagenesis in Fab 57P, specific for peptide 134 to 151 of the coat protein of tobacco mosaic virus. The 13 single mutants were analysed by Western blot to determine the effect of interface modifications on Fab expression. The kinetic rate constants of peptide-Fab mutant interactions were measured using the biosensor technology. Alanine replacements did not prevent assembly of the mutated Fabs and led to a modification of their binding properties in every case. Twelve of the 13 target residues correspond to homologous positions in the VL and VH domains, which have similar folds. Mutation at homologous positions mostly had different effects on antigen binding affinity. The replacement of bulky side-chains had the most drastic effect on binding. When smaller side-chains were replaced by alanine, the binding properties of Fab mutants differed slightly (by less than a factor of two), but significantly from that of Fab 57P. Modification of some of these residues, which are located 9 to 12 A away from the base of CDR loops, is unlikely to alter loop conformation. They may affect antigen binding indirectly by influencing the relative position of the VL and VH domains. Our results demonstrate that residues situated at the VL-VH interface and which are remote from the paratope are able to influence the antigen binding properties of antibodies.

Amino Acid Sequence↗

PubMed1995

Analysis of cyclosporin interactions with antibodies and cyclophilin using the BIAcore.

The immunosuppressive cyclic undecapeptide cyclosporin A (CS) exists in various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin or with a monoclonal antibody. Differences in the ability of CS and its analogs to bind to antibody or cyclophilin have been measured using the BIAcore. These experiments suggest that the rate-limiting step of complex formation is determined by the interconversion between different CS conformers existing in solution. The contribution to antibody binding of individual atomic groups of CS was evaluated by measuring the equilibrium affinity constants of analogs with the BIAcore. When the binding data were analyzed in terms of the known crystallographic structure of the CS/Fab complex, it could be shown that modifications of CS residues located in the central part of the binding site drastically affect affinity, while modifications of residues located at the periphery are more easily accommodated.

Amino Acid Isomerases↗

PubMed1994

Interaction of cyclosporin A and two cyclosporin analogs with cyclophilin: relationship between structure and binding.

The immunosuppressant drug cyclosporin A exists as various conformers in water. Up to 1 h is needed to reach maximum complex formation after mixing the drug with its receptor, cyclophilin, or an antibody, indicating that only a fraction of the conformers in aqueous solution adopts a conformation suitable for binding. In the present study we compare the binding behavior of cyclosporin to that of two analogs, using a biosensor instrument (BIAcore, Pharmacia). The amount of complex formation was measured as a function of time after adding the peptides to cyclophilin. The equilibrium affinity constants of cyclophilin for these analogs have been measured. The slow binding of cyclosporin to cyclophilin compared to the instant binding of the cyclosporin analogs supports the hypothesis that cyclophilin recognizes a well defined conformation of cyclosporin that exists in water prior to binding.

Amino Acid Isomerases↗

PubMed1994

Conformational polymorphism of cyclosporin A.

BACKGROUND: Cyclosporin A (CsA) is a cyclic undecapeptide fungal metabolite with immunosuppressive properties, widely used in transplant surgery. It forms a tight complex with the ubiquitous 18 kDa cytosolic protein cyclophilin A (CypA). The conformation of CsA in this complex, as studied by NMR or X-ray crystallography, is very different from that of free CsA. Another, different conformation of CsA has been found in a complex with an antibody fragment (Fab). RESULTS: A detailed comparison of the conformations of experimentally determined structures of protein-bound CsA is presented. The X-ray and NMR structures of CsA-CypA complexes are similar. The Fab-bound conformation of CsA, as determined by X-ray crystallography, is significantly different from the cyclophilin-bound conformation. The protein-CsA interactions in both the Fab and CypA complexes involve five hydrogen bonds, and the buried CsA surface areas are 395 A2 and 300 A2, respectively. However, the CsA-protein interactions involve rather different side chain contacts in the two complexes. CONCLUSIONS: The structural results presented here are consistent with CypA recognizing and binding a population of CsA molecules which are in the required CypA-binding conformation. In contrast, the X-ray structures of the Fab complex with CsA suggest that in this case there is mutual adaptation of both receptor and ligand during complex formation.

Amino Acid Isomerases↗

PubMed1994

Structure-activity relationships for the interaction between cyclosporin A derivatives and the Fab fragment of a monoclonal antibody.

The crystallographic structure of a complex between cyclosporin A and the Fab fragment of monoclonal antibody R45-45-11 has been solved to 2.65 A resolution (Altschuh et al., 1992a, Science 256, 92; Vix et al., 1993, Proteins 15, 339), yielding a precise three-dimensional picture of interacting surfaces. In order to evaluate the contribution of observed contacts to the energy of interaction, we have measured the effect on binding affinity of minor chemical modifications of CS. The equilibrium binding constant of the Fab fragment for a set of cyclosporin analogs was obtained by measuring in a biosensor instrument the dependence of complex formation on Fab concentration, at constant analog concentrations. Data were analysed using Scatchard plots. Differences in binding energy resulting from cyclosporin modifications discriminated between two types of contact areas. The first type displays adaptability to structural modifications of cyclosporin at the cost of a small decrease in binding energy, and contacting residues in the antibody form the periphery of the combining site. The second type does not accommodate structural changes and corresponds in cyclosporin to three residues whose modifications drastically decrease binding energy with the antibody. The corresponding contact residues in the antibody form the core of the antibody combining site.

Amino Acid Sequence↗

PubMed1994

Modulation of the enzymatic activity of papain by interdomain residues remote from the active site.

The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains. This topology suggests a possible relationship between relative domain organization and catalytic mechanism. The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues. Most modifications had no effect on enzyme activity. However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site. The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair. This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered. Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.

Base Sequence↗

PubMed1993

Interaction of cyclosporin A with an Fab fragment or cyclophilin. Affinity measurements and time-dependent changes in binding.

Different conformers of the immunosuppressant cyclosporin A have been observed in structural studies of the isolated molecule and of its complex with cyclophilin or with an Fab fragment. The factors that control this conformational change are not well understood. Variations in the amount of complex formed with cyclophilin or with the antibody were measured as a function of time after adding cyclosporin to the proteins, using the Pharmacia BIAcore biosensor instrument. Up to 1 hour was needed to reach maximum complex formation in solution, which is likely to reflect the time needed for a conformational transition of cyclosporin. The equilibrium affinity constant of both proteins for cyclosporin has been measured.

Amino Acid Isomerases↗

PubMed1993

Epitope analysis using kinetic measurements of antibody binding to synthetic peptides presenting single amino acid substitutions.

The fine modulation of peptide-antibody interactions was investigated with anti-peptide monoclonal antibodies recognizing peptide 125-136 of the coat protein of tobacco mosaic virus. Nine synthetic peptides presenting single amino acid substitutions were selected for detailed analysis on the basis of their reactivity in ELISA. Kinetic measurements of the binding of four antibodies to these peptides performed with a biosensor instrument (BIAcore, Pharmacia) were used to quantify the contribution of individual residues to antibody binding. The results showed that even conservative exchanges of some residues in the epitope resulted in a small but significant decrease of the equilibrium affinity constant due mostly to a higher dissociation rate constant of the monoclonal antibodies. Two amino acid residues directly adjacent to the epitope, which appeared to play no role when tested by ELISA, were shown to influence the kinetics of binding. These data should be useful for computer modelling of the peptide-antibody interactions.

Amino Acid Sequence↗

PubMed1993

Crystallographic analysis of the interaction between cyclosporin A and the Fab fragment of a monoclonal antibody.

The structure of the complex between cyclosporin A and the Fab fragment of a monoclonal antibody has been established by crystallographic analysis to 2.65 A resolution. The structure has been solved by molecular replacement using a composite Fab model. The current R-factor after refinement is 0.179 between 8 and 2.65 A resolution. The antibody is one among three known structures with long H3 loops. This loop conformation is observed for the first time in the presence of the antigen. Residues from all six hypervariable loops interact with cyclosporin A. However, the 17 residues long loop H3 is the main contributor to the buried combining site area and to the van der Waals contacts made with cyclosporin A, with 52 and 63%, respectively, of the total contribution.

Amino Acid Sequence↗

PubMed1993

Monoclonal antipeptide antibodies: affinity and kinetic rate constants measured for the peptide and the cognate protein using a biosensor technology.

The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.

Amino Acid Sequence↗

PubMed1993

Tobacco mosaic virus: a model antigen to study virus-antibody interactions.

For more than 50 years, tobacco mosaic virus (TMV) has been used as a model system for studying various aspects of virus-antibody interactions. Distinct epitopes called neotopes and cryptotopes have been identified in intact TMV particles and dissociated viral protein respectively and a correlation has been found to exist between the location of continuous epitopes and the extent of segmental mobility along the viral polypeptide chain. The occurrence of bivalent antibody binding was shown to influence the observed affinity of TMV antibodies and kinetic measurements of antibody binding to viral peptides made it possible to analyze the mechanism of binding of monoclonal antibodies. It seems likely that the TMV model will continue to yield a rich harvest of immunochemical data relevant to many viral systems.

Antibodies, Monoclonal↗