Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N- and C-terminal low affinity contributing regions.

The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19-32, 45-54, 129-138, 145-164, 161-178 and 191-210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10(-6)-10(-7) M, except for hcTnI [39-58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19-32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19-32 may correspond to the minimal sequence of the extension which could switch between the N- and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca(2+)-dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164-178 and 191-210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.

Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display. Role of sequences flanking thebinding motif.

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage-displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y-TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15-mer synthetic peptides derived from the phage-selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7-26 nM range. These affinities represented, however, only 1.9-7. 5% of the affinity of the 15-mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low-affinity peptide selected by phage-display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N- or the C-terminal sequence of the peptide epitope to the Y-TEPK motif of a low-affinity peptide selected by phage-display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high- or low-affinity peptides will be obtained from phage display.

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.

Antigenic definition of cardiac troponin I.

The presence of cardiac troponin I in the serum is now considered as one of the most specific biochemical markers of acute myocardial infarction. To improve the knowledge of the antigenic properties of cardiac Troponin I, a set of monoclonal antibodies and polyclonal antibodies against human cardiac troponin I has been tested with overlapping peptides covering the cardiac troponin I sequence. The results indicate that N-terminal and C-terminal cardiac troponin I regions were most often recognized by poly- and monoclonal antibodies. These observations are valuable for choosing the best combination of monoclonal antibodies to set up new immunoassays to detect serum cardiac troponin I earlier after myocardial damage, to understand better which forms are released, and finally to propose appropriate cardiac troponin I standards.

Human cardiac troponin I: precise identification of antigenic epitopes and prediction of secondary structure.

The presence of human cardiac troponin I (hcTnI) in serum is considered to be a highly specific biochemical marker of acute myocardial infarction. To better understand the antigenic properties of hcTnI, a set of 68 overlapping peptides covering the complete amino acid sequence of hcTnI was prepared and used in epitope mapping experiments. All 16 anti-hcTnI monoclonal antibodies tested were found to recognize a peptide epitope, indicating that recognition by anti-hcTnI monoclonal antibodies was not dependent on the tertiary structure of the protein. Furthermore, the peptide reactivity with anti-hcTnI polyclonal antibodies indicated that most of the sequence of the protein was antigenic; in particular, the N- and C-terminal extremities were found to be the strongest antigenic regions. By using accurate secondary structure prediction methods, hcTnI was found to be an all-alpha type protein, with five regions predicted as helices. Matching the results of the epitope analysis with the structural prediction led us to the view that hcTnI is not a globular protein but probably adopts an extended conformation, allowing a large part of the amino acid sequence of this molecule to be recognized by the immune system. This improved knowledge of the antigenic and structural properties of hcTnI may help in developing new antibodies and immunoassays for use in diagnosing myocardial infarction.

Variation of bcl-2 expression in breast ducts and lobules in relation to plasma progesterone levels: overexpression and absence of variation in fibroadenomas.

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl-2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl-2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl-2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean = 10.1, quantitative immunochemistry score (QIC) arbitrary units (AU), n = 19], than in normal lobules (mean = 5.1 AU, n = 43, P = 7 x 10(-5). bcl-2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P = 1.8 x 10(-2) and P = 1.7 x 10(-2), respectively). This was further supported by a statistical link (P = 5 x 10(-3) between high levels of circulating progesterone and weak bcl-2 staining in lobules and ducts. This progesterone-dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl-2 expression and circulating levels of oestradiol, and follicle-stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl-2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins.

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 x 10(-7) to 6.7 x 10(-8) M-1 for the soluble form of 9 lysozyme-binding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.