Synthetic Peptide Approach Research Articles

Localization of an immune functional site on staphylococcal enterotoxin A using the synthetic peptide approach.

Abstract Using the synthetic peptide approach, we have identified a part of the staphylococcal enterotoxin A (SEA) molecule that is responsible for stimulation of T cell proliferation and induction of the lymphokine IFN-gamma. Peptides were synthesized corresponding to amino acids 1 to 27, SEA(1-27), and 28 to 45, SEA(28-45). Both peptides were tested for direct competition with SEA for blockage of SEA induced proliferation and production of IFN-gamma by T cells. Further, antibodies were produced to the peptides and tested for their ability to bind to SEA and block SEA function. SEA (1-27), but not SEA (28-45), blocked proliferation of human peripheral T cells and induction of IFN-gamma by the T cell line, L12-R4. The inhibitory effects were specific, because SEA (1-27) did not inhibit the induction of T cell proliferation by the mitogen PHA. Consistent with the direct inhibition of function, antibodies to SEA (1-27), but not SEA (28-45), neutralized the mitogenic activity of SEA on human PBL. The data suggest that a functional site on SEA that is responsible for its modulation of T cell function involves the N-terminal 27 amino acids. Residues 1 to 27 of SEA could potentially interact at either the level of the TCR or may block the proposed binding of SEA to class II MHC Ag, based on recent data showing that these molecules are involved in SEA-induced proliferation.

Topographic Analysis of Human Epidermal Growth Factor by Monospecific Antibodies and Synthetic Peptides

The mode of interaction between human epidermal growth factor (hEGF) and its receptor has been investigated by immunochemical studies and a synthetic peptide approach. Two types of monoclonal and five different monospecific polyclonal antibodies against hEGF have been prepared, whose epitopes are regions 1-13, 13-32, 33-53, 33-43, 22-32, and discontinuous sequences of hEGF. Antibody against 22-32 (Type I) and antibody against 33-53 (PRE 4) inhibited the binding of 125I-hEGF to membrane receptor on A 431 cells more markedly than the other antibodies. When hEGF was bound to the receptor, only antibody against 13-32 (PRE 2) could bind to hEGF-receptor complex whereas antibody against 22-32 (Type I) could not. These data suggest that region 13-20 is exposed outside during receptor-binding and both region 22-32 and region 33-53 contact the hEGF receptor. The activity of synthetic peptides corresponding to the amino acid residues 1-13, 13-32, 33-53, 13-20, 22-32, and 33-43 of hEGF was also examined. Out of the six peptides, only 13-32 stimulated DNA synthesis of BALB 3T3 cells. The activity was approximately 1/10(6) of that of intact hEGF. All of these data suggest that region 22-32 is responsible for binding to the receptor for signal transduction and region 33-53 binds to the receptor to stabilize the ligand-receptor interaction. This dual binding model fits in well with the three-dimensional hEGF structure deduced from NMR spectra.

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells.

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.

Induction of polyclonal and monoclonal antibody responses to cholera toxin by the synthetic peptide approach

The induction of an antibody response to cholera toxin (CT) was studied by using the synthetic peptide approach. Two peptides, corresponding to the amino acid sequences from residues 57 to 69 (CTBP 1) and 47 to 60 (CTBP 2) of the cholera toxin B subunit, were synthesized by the solid-phase method. These peptides were primarily chosen on the basis of their hydrophilicity and sequence identity with the B subunit of E. coli toxin (LT h). Synthesized peptides were coupled to carrier proteins through additional cysteine residues at the carboxyl (CTBP 1) or amino terminal ends (CTBP 2). Rabbit antisera to the peptide-carrier conjugates were found to react with the free peptides as well as intact CT, its B subunit and LT h as determined by the conventional enzyme-linked immunosorbent assay (ELISA). On the other hand, anti-peptide sera failed to react with CT and LT h in GM 1 (ganglioside)—ELISA, thereby suggesting the possible involvement of ctbp 1 and CTBP 2 peptide regions of the toxin molecule in GM 1 receptor binding. Both anti-peptide sera possessed rather weak toxin neutralizing activity in the rabbit ileal loop assay. However, such activity was statistically significant (0.02 < P < 0.05) only in the case of anti-CTBP 2 serum. Similar results were also obtained with mouse polyclonal anti-peptide sera. Ten mouse monoclonal antibodies were obtained against the CTBP 1 peptide, five of which reacted to CT, the B subunit and LT h in ELISA. Interestingly, one monoclonal showed strong reactivity against CT and LT h although it reacted very weakly against the immunizing peptide CTBP 1. It appears that the immunizing peptide probably exists in multiple conformers in the conjugated form, some of which may mimic more closely its structural features in the intact protein than in the free state. Results obtained in this study suggest that synthetic peptides can serve as useful probes for the structural analysis of CT or related toxins and may be useful in vaccine development.

Epitope and functional specificity of monoclonal antibodies to mouse interferon-gamma: the synthetic peptide approach.

Spleen cells from hamsters immunized with recombinant mouse interferon-gamma (IFN-gamma) were fused with mouse myeloma cells, resulting in the production of four anti-IFN-gamma monoclonal antibodies. Binding of 125I-IFN-gamma by these protein A-bound antibodies was specifically blocked by cold IFN-gamma. Binding by three of these antibodies was also blocked by a synthetic peptide corresponding to the N-terminal 1-39 amino acids of IFN-gamma, whereas a corresponding C-terminal (95-133) peptide had no effect on binding. The N-terminal specificity of these three antibodies was confirmed by their specific binding of 125I-N-terminal (1-39) peptide. One of the N-terminal specific monoclonal antibodies inhibited both antiviral and macrophage priming (for tumor cell killing) activities of IFN-gamma, whereas the other two had no effect on either biologic function. The selectivity of the inhibition of IFN-gamma function was not due to a differential ability of the N-terminal specific antibodies to bind IFN-gamma. Blocking experiments with cold IFN-gamma and N-terminal peptide suggest that the epitope specificities of the monoclonal antibodies could be determined by the conformational or topographic structure of IFN-gamma. An exact determination of the epitope specificity of the monoclonal antibody that inhibited IFN-gamma function could provide insight into the structural basis for the role of the N-terminal domain in the biologic function of IFN-gamma. Polyclonal antibodies to either the N-terminal or the C-terminal peptides also inhibited both the antiviral and the macrophage-priming activities of IFN-gamma. All of the antibodies that inhibited IFN-gamma function also blocked binding of IFN-gamma to membrane receptor on cells, whereas antibodies that did not block function also did not inhibit binding. The data suggest that both the N-terminal and the C-terminal domains of IFN-gamma play an important role in its antiviral and macrophage-priming functions, possibly in a cooperative manner.

The value of synthetic peptides as vaccines for eliciting T-cell immunity.

As our understanding of T-cell activation has increased at the molecular level, several investigators have begun to discuss the idea of using synthetic peptides as vaccines for inducing T-cell immunity (LERNER 1984; LAVER and AIR 1985). My view as to the value of such an approach is rather pessimistic. In this paper I will briefly outline our current understanding of antigen recognition by T cells and then discuss in detail what I see to be the major pitfalls in the peptide approach to Vaccine development. I do not mean to imply in this discussion that developing vaccines to induce T-cell immunity represents a fruitless endeavor. Rather, I wish to stress that the synthetic peptide approach may not be the most rational one to choose or the most cost-effective at the present time.KeywordsMajor Histocompatibility ComplexSynthetic PeptideAllelic FormMajor Histocompatibility Complex HaplotypeHelper DeterminantThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synthetic peptide approach to the analysis of kinase activities of avian EGF receptor and v-erbB protein

Analysis of the structure and function of a protein such as the epidermal growth factor receptor is facilitated by the use of antibodies directed against discrete portions of the protein. Here, we describe the characterization and use of antibodies directed against synthetic peptides corresponding to specific portions of the epidermal growth factor receptor and/or v-erbB protein. In particular, one useful antiserum has allowed us to compare the protein kinase activities of the epidermal growth factor receptor and the v-erbB proteins and to conclude that the v-erbB protein is a protein-tyrosine specific kinase as is its homologue the avian epidermal growth factor receptor.