Integrin α5ß1 and p53 convergent pathways in the control of anti-apoptotic proteins PEA-15 and survivin in high-grade glioma.

Integrin α5ß1 expression is correlated with a worse prognosis in high-grade glioma. We previously unraveled a negative crosstalk between integrin α5ß1 and p53 pathway, which was proposed to be part of the resistance of glioblastoma to chemotherapies. The restoration of p53 tumor-suppressor function is under intensive investigations for cancer therapy. However, p53-dependent apoptosis is not always achieved by p53-reactivating compounds such as Nutlin-3a, although full transcriptional activity of p53 could be obtained. Here we investigated whether integrin α5ß1 functional inhibition or repression could sensitize glioma cells to Nutlin-3a-induced p53-dependent apoptosis. We discovered that α5ß1 integrin-specific blocking antibodies or small RGD-like antagonists in association with Nutlin-3a triggered a caspase (Casp) 8/Casp 3-dependent strong apoptosis in glioma cells expressing a functional p53. We deciphered the molecular mechanisms involved and we showed the crucial role of two anti-apoptotic proteins, phosphoprotein enriched in astrocytes 15 (PEA-15) and survivin in glioma cell apoptotic outcome. PEA-15 is under α5ß1 integrin/AKT (protein kinase B) control and survivin is a p53-repressed target. Moreover, interconnections between integrin and p53 pathways were revealed. Indeed PEA-15 repression by specific small-interfering RNA (siRNA)-activated p53 pathway to repress survivin and conversely survivin repression by specific siRNA decreased α5ß1 integrin expression. This pro-apoptotic loop could be generalized to several glioma cell lines, whatever their p53 status, inasmuch PEA-15 and survivin protein levels were decreased. Our findings identify a novel mechanism whereby inhibition of α5ß1 integrin and activation of p53 modulates two anti-apoptotic proteins crucially involved in the apoptotic answer of glioma cells. Importantly, our results suggest that high-grade glioma expressing high level of α5ß1 integrin may benefit from associated therapies including integrin antagonists and repressors of survivin expression.

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.

Recognition of peptides by antibodies and investigations of affinity using biosensor technology.

The study of peptide-antibody interactions has many applications in biology and medicine. Synthetic peptides corresponding to single protein epitopes are used instead of intact proteins as reagents for the diagnosis of viral and autoimmune diseases. Furthermore, antibodies raised against peptides are useful reagents for isolating and characterizing gene products. In this review, methods for analysing the molecular basis of peptide-antibody interactions are described, such as amino acid replacement studies, X-ray crystallography of peptide-antibody complexes and biosensor technology based on surface plasmon resonance. The importance of peptide conformation in antibody recognition is discussed, and the antigenic reactivity of epitopes in synthetic peptides and in cognate, intact proteins is compared.

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.

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.

Production of fluorescent single-chain antibody fragments in Escherichia coli.

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.

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.

Predicting the kinetics of peptide-antibody interactions using a multivariate experimental design of sequence and chemical space.

A multivariate approach involving modifications in peptide sequence and chemical buffer medium was used as an attempt to predict the kinetics of peptide-antibody interactions. Using a BIACORE system the kinetic parameters of the interaction of Fab 57P with 18 peptide analogues of an epitope of tobacco mosaic virus protein were characterized in 20 buffers of various pH values and containing different chemical additives (NaCl, urea, EDTA, KSCN and DMSO). For multivariate peptide design, three amino acid positions were selected because their modification was known to moderately affect binding, without abolishing it entirely. Predictive mathematical models were developed which related kinetic parameters (k(a) or k(d)) measured in standard buffer to the amino acid sequence of the antigen. ZZ-scales and a helix-forming-tendency (HFT) scale were used as descriptors of the physico-chemical properties of amino acids in the peptide antigen. These mathematical models had good predictive power (Q(2) = 0.49 for k(a), Q(2) = 0.73 for k(d)). For the non-essential residues under study, HFT and charge were found to be the most important factors that influenced the activity. Experiments in 19 buffers were performed to assess the sensitivity of the interactions to buffer composition. The presence of urea, DMSO and NaCl in the buffer influenced binding properties, while change in pH and the presence of EDTA and KSCN had no effect. The chemical sensitivity fingerprints were different for the various peptides. The results indicate that multivariate experimental design and mathematical modeling can be applied to the prediction of interaction kinetics.

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context.

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.