Helga Wessner

Changing the Antigen Binding Specificity by Single Point Mutations of an Anti-p24 (HIV-1) Antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr32Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe94Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe94Ala up to the level of the e-pep affinity to the wild-type scFv.

Structural base of the interaction of a monoclonal antibody against p24 of HIV-1 with its peptide epitope

The interaction of a murine monoclonal antibody (CB 4-1) against the core protein p24 of HIV-1 with its peptide antigen was studied in detail. The amino acid sequence of the variable regions of the heavy and light chain as derived from DNA sequencing was used to model the structure of the antigen binding region on the basis of reported Fab structures from the Brookhaven Protein Data Base. A linear peptide epitope responsible for the p24 binding to the antibody was determined by peptide scan. Subsequent N- and C-terminal truncation of the corresponding sequence region as well as amino acid substitutions were performed to recognize the epitope and the amino acid residues critical for antibody binding. These data were used to derive a structural model of the peptide-antibody interaction.

The structure of the anti-c-myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops

The X‐ray structure of the Fab fragment from the anti‐c‐myc antibody 9E10 was determined both as complex with its epitope peptide and for the free Fab. In the complex, two Fab molecules adopt an unusual head to head orientation with the epitope peptide arranged between them. In contrast, the free Fab forms a dimer with different orientation. In the Fab/peptide complex the peptide is bound to one of the two Fabs at the “back” of its extended CDR H3, in a cleft with CDR H1, thus forming a short, three‐stranded antiparallel β‐sheet. The N‐ and C‐terminal parts of the peptide are also in contact with the neighboring Fab fragment. Comparison between the CDR H3s of the two Fab molecules in complex with the peptide and those from the free Fab reveals high flexibility of this loop. This structural feature is in line with thermodynamic data from isothermic titration calorimetry. Proteins 2008.

Phage display screening for peptidic chitinase inhibitors.

A phage display library with disulfide‐cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose‐bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms. Copyright

Defining the immunoreactive epitope for the monoclonal anti-human glutathione peroxidase-4 antibody anti-hGPx4 Mab63-1

Glutathione peroxidases (GPx) form a heterogeneous enzyme family and GPx4-isoforms have been implicated in anti-oxidative defense, brain development, neuroinjury and sperm maturation. In humans seven GPx isoforms (GPx1-GPx7) can be separated. To selectively quantify the expression of GPx4-isoforms we have raised a monoclonal antibody (anti-hGPx4 Mab63-1) against the pure recombinant Sec46Cys mutant of human cytosolic GPx4 and used it for immunoblotting, immunoprecipitation and immunohistochemistry. The antibody recognizes human GPx4, its mouse ortholog but neither reacted with rat GPx4 nor other human GPx-isoforms. Sequence alignment of human and rat GPx4 proteins indicated three different amino acids (S18, F35, K99 in humans, A18, C35, R99 in rats) and a S18A exchange in the human enzyme completely abolished immunoreactivity. To further characterize the immunological epitope we synthesized a set of 12-mer peptides flanking S18* of human GPx4 and found that the sequence SMHEFS*AKDIDG exhibited strongest immunoreactivity. Substitution analysis and peptide length variation narrowed down the essential epitope to FS*AKDI and indicated that most mutations in this region strongly impaired immunoreactivity. In silico blast searches of public protein databases failed to identify proteins with potential immunoreactivity suggesting that the antibody exhibits a high specificity for human and mouse GPx4 and may not cross-react with unrelated proteins.