Chemotaxis induced by SXWS tetrapeptides in Tetrahymena--overlapping chemotactic effects of SXWS sequences and their identical amino acids.

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position 'X' of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS-SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent-exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer-assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the 'X' position indicate that member 'X' of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane.

Heteroclitic recognition of combinatorial TX1TX2T peptide mixtures by mucin-2 protein specific monoclonal antibody.

The mucin-2 (MUC2) glycoprotein secreted by the epithelial cells of human colon may be abnormally under-glycosylated in the case of cancer. Monoclonal antibody (mAb) 994 raised against the immunogenic part of the protein core, recognizes malignant human colon tissues as well as pentapeptides with TX1TX2T motif present in MUC2. Using a combinatorial approach and ELISA experiments it was found that mAb 994 is able to recognize peptides of the sub-library TQTX2T very strongly, and to some extent also peptides from TETX2T, TLTX2T and TVTX2T sub-libraries. Binding studies with peptides corresponding to the TQTX2T and TETX2T sub-libraries showed that mAb 994 recognized only six peptides (IC50 = 9-208 micromol dm(-3)) from the 19 compounds of the TQTX2T sub-library and only three peptides (IC50 = 3500-16700 micromol dm(-3)) from the 'second-best' TETX2T sub-library. The most pronounced mAb binding occurred when Gln was in position X1 and it was much weaker in the case of Glu, Val or Leu. As for X2 amino acids, the presence of Pro, Ala can provide a strong, while Tyr, Trp, Phe and Ser a weaker, peptide-antibody interaction. Data from this study suggest that pentapeptide TQTPT, whose sequence is present in the native protein, is bound most strongly. However, almost identical binding properties were observed with peptide TQTAT, whose sequence is not present in the protein. Apart from this, some other 'heteroclitic' peptides were found with a different rank in the binding-hierarchy. Based on these peptides artificial compounds can be prepared as potential candidates for vaccine development. Results of this study also provide a rationale for understanding the molecular background of the heteroclitic nature of the MUC2 protein core specific mAb 994.

Characterization of chemotactic ability of peptides containing N-formyl-methionyl residues in Tetrahymena fMLP as a targeting ligand.

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C-terminal f-Met and N-terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f-MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f-Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis-activating processes.

Chemotactic activity of oligopeptides containing an EWS motif on Tetrahymena pyriformis: the effect of amidation of the C-terminal residue.

Chemotactic properties of 3-7-mer peptides containing an EWS motive and their peptide amides synthesized and characterized by us were investigated in Tetrahymena pyriformis GL model. Analysis of the peptide acids shows that SEWS possesses exceptionally strong (660%+/-21; 430%+/-18) chemoattractant ability at 10(-12) and 10(-11) m respectively. The shorter peptide (EWS) possesses chemorepellent activity, while longer peptides display neutral (WSEWS) or moderate chemoattractant (EWSEWS and GEWSEWS) chemotactic ability. Amidation of the C-terminus can significantly modify the character of peptides: it points to the conclusion that a free alpha-COOH group at this position is required for the high efficiency of SEWS, while in the shorter (EWS) and longer peptides (WSEWS and EWSEWS) amidation can result in chemoattractant ligands. Evaluation of the structure-function relationship of these compounds establishes the significance of Glu (E) with its high surface-exposed area and negatively-charged side chain. The high discriminative ability and good chemotactic responsiveness of Tetrahymena support the theory that a chemotactic signalling mechanism working in higher levels of phylogeny is a well conserved and inducible one even in protozoa.

Characterisation of combinatorial libraries of mucin-2 antigen peptides by high-resolution mass spectrometry.

An epitope motif, TX(1)TX(2)T, of mucin-2 glycoprotein was identified by means of a mucin-2-specific monoclonal antibody, mAb 994, raised against a synthetic mucin-derived 15-mer peptide conjugate. For determination of the epitope sequence recognised with highest affinity by mAb 994, a combinatorial approach was applied using the portioning-mixing technique excluding Cys. Antibody binding of libraries was most profound when Gln was at the X(1) position. Analytical characterisation of the TQTX(2)T library was conducted by amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and electrospray ionisation Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometric methods. Control libraries were prepared by mixing 19 individual peptides corresponding to the TQTX(2)T sequence. Thus, mixtures of 6, 10 and 19 pentapeptides were analysed and compared with the combinatorial mixture. MALDI-TOFMS was able to detect only partially the components in the 6- and 10-member mixtures, but failed to characterise a more complex 19-member mixture. In contrast, ESI-FTICRMS resolved all mixtures of higher complexity and provided direct identification at monoisotopic resolution, such as for a peptide library containing 'isobaric' lysine and glutamine (Delta m = 0.0364 Da). The results of this study suggest that ESI-FTICRMS is a powerful tool for characterisation of combinatorial peptide libraries of higher complexity.

Synthesis of oligopeptides with the sequence SXWS and their chemotactic effects on a ciliated protozoan Tetrahymena pyriformis.

In this paper, the solid phase synthesis and chemical characterization of members of an SXWS sub-library (SAWS, SDWS and SKWS) as well as the comparison of their chemotactic properties with those of SEWS, which exhibits a prominent effect at 10(-12) M on a ciliated protozoan, Tetrahymena pyriformis, are described. We found that the chemotaxis of cells induced with the SXWS peptides varied according to the nature of the amino acid residue (Ala, Asp, Lys) in position X. The chemotactic activity of SEWS was not surpassed by any of three new tetrapeptides, although SAWS was also chemoattractant. Interestingly, SDWS, with an acidic side chain at position X, could not elicit any chemotactic response. SKWS, however, showed mild but significant chemorepellent activity over a wide concentration range. Chemotactic selection studies showed that the two chemoattractant peptides (SAWS and SEWS) had an expressed ability to select high-responder offspring cell populations. Peptides with neutral (SDWS) or chemorepellent (SKWS) properties were not able to select such subpopulations from the mixed cultures of Tetrahymena, indicating that the chemotactic response elicited by SXWS peptides is ligand-specific. For ligand-binding experiments N-terminally labelled fluorescent derivatives of SXWS peptides were prepared. applying [4-[7-hydroxycoumaryl]]acetic acid (Hca-OH) or 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) as markers. Hca-OH was introduced using an active ester technique as the last step of SPPS, or after cleavage in solution. The oxazolone naOx-OEt reacted with the amino group of the peptide by liberation of EtOH. The binding characteristics of fixed Tetrahymena cells with the naOx-labelled peptides showed good correlation between binding profiles and chemotactic responsiveness (SEWS > SAWS > SDWS - SKWS). A similar binding pattern was observed in the case of Hca-peptides (SEWS > SAWS > SDWS). Hca-SKWS, however, bound remarkably to the cell surface. The binding activity of the Hca-peptides was less pronounced than that of the naOx-peptides, indicating the importance of the fluorophores applied.