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Chaperonin 10 of M. tuberculosis conferred partial or total protection against generalized foot-and-mouth disease (FMD) in guinea-pigs challenged with O1 Lausanne FMD virus. Chaperonin 10-immunized animals mounted an antibody response to the protein, one epitope of which was found in the C-terminal half. A similar recognition pattern was observed in FMD-convalescent guinea-pigs, swine and cattle. Anti-chaperonin 10 sera showed antiviral activity against FMDV-infected BHK-21 cells. There was strong evidence that early after infection these cells actively secrete their histones and that antisera to the chaperonin recognize them. The same antisera reacted with purified histones in immunoblotting. Most important, exogenously added histones abrogated the anti-viral activity of the antiserum and an anti-histone monoclonal antibody had strong antiviral activity against FMDV-infected BHK-21 cells. These results are consistent with previous reports on displacement of histones from the nuclear compartment and immune recognition of self-histones after viral infections. On the whole, they indicate that M. tuberculosis chaperonin 10 enables the immune system to react against early abnormalities of virus-infected cells; this is accomplished by antibody cross-reacting with histones released during virus infection.

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The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoelectricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required, (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters, (iii) Very little manipulation is needed, and each focusing requires 3-5h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

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We have previously described the conditions by which peptide synthesis by the solid-phase fragment condensation approach can be carried out using crown ethers as non-covalent protection for the N alpha-amino group. Here we demonstrate that the procedure can be extended to large, partially protected peptide fragments possessing free Lys and/or Arg residues. The first step was to ensure that complex formation on the side chain of amino acids was not detrimental to the methodology and exhibited the same solubility and coupling properties as N alpha-complexed peptides. Thus, a model hexapeptide was synthesized using Fmoc chemistry containing Lys and Arg residues, which, when complexed with 18-Crown-6, was readily soluble in DCM and coupled quantitatively to a resin-bound tetrapeptide. Two tripeptides were then prepared, one containing a free Ser residue, the other free Tyr, to examine the possible occurrence of side reactions. After coupling using standard conditions only the former tripeptide exhibited the formation of the O-acylation by-product (5%). Another model hexapeptide containing Lys, Tyr, Ser and Asp protected with a TFA-stable adamantyl group was complexed with 18-Crown-6 and coupled to the resin-bound tetrapeptide with near quantitative yield. Extending the length of the peptide to 21 and 40 residues, which represent sequences Gly52 to Leu72 (21-mer) and Pro33 to Leu72 (40-mer) from Rattus norvegicus chaperonin 10 protein, respectively, resulted in partially protected fragments that were readily soluble in water, thus enabling purification by RP-HPLC. Complexation with 18-Crown-6 gave two highly soluble products that coupled to resin-board tetramer with 68% and 50% coupling efficiencies for the 21-mer and 40-mer, respectively. Treatment with 1% DIEA solutions followed by acidolytic cleavage and purification of the major product confirmed that the correct product has been formed, when analysed by amino acid analysis and ESI-MS. These results served to extend the methodology of non-covalent protection of large partially protected peptide fragments for the stepwise fragment condensation of polypeptides.

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The heat shock protein, hsp10, is an abundant protein in Mycobacterium tuberculosis (Mtb), its nucleotide sequence encoding a protein of 99 amino acids with a molecular mass of 10.7 kD. This sequence is phylogenetically conserved, being represented by the GroES homologue of Escherichia coli. Hsp10 and GroES are members of the chaperonin 10 family of molecular chaperones, and GroEs is necessary for the optimal activity of GroEL, a member of the chaperonin 60 family and the E. coli homologue of mycobacterial hsp65. Since hsp65 has been implicated in both experimental and human rheumatoid arthritis, we aimed to assess the immunomodulatory effects of its co-chaperonin, hsp10, in experimental arthritis. Our results show that an aqueous solution of a mycobacterial hsp10 delayed the onset and severity of adjuvant-induced arthritis in rodents when administered after disease induction but before joint involvement occurred. This biological activity was specific for the hsp10 of Mtb, since neither GroES not the rat homologue was effective. Using synthetic hsp10 fragments, the activity was localized to the N-terminal region of the molecule. Assessment of circulating antibody levels to mycobacterial hsp10 and hsp65 indicated that all arthritic rats had increased titres to both hsp10 and hsp65: hsp10-treated rats showed further elevation of this humoral response not only to hsp10 but also to hsp65 when compared with the untreated arthritic control. This is the first report of the immunomodulatory activity of mycobacterial hsp10 in experimental arthritis, and exhibits a potential role for this co-chaperonin in pathophysiological situations.

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The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity. Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel beta-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins.

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The peptide corresponding to the C-terminal half of M.tuberculosis hsp10 was synthesised based on the prediction that it might represent an independent structural region of the protein. This hypothesis was confirmed by aggregation and CD studies using this peptide and longer sequences of the protein. The peptide shares about 40-50% sequence homology with alpha 2 and beta 1 chains of MHC class I and II antigens. This and the CD results which indicated that the peptide at acidic pHs folds into an anti-parallel beta-sheet were used to generate a 3D model which has the same "W" fold contained in the MHC peptide binding groove. These data suggest that the hypothesis of molecular mimicry proposed to be one of the mechanisms which triggers autoimmune diseases may be extended to hsp10 proteins. Furthermore the suggested evolutionary relationship between hsp's and MHC antigens may find support from these data.

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Recently, the sequence of mitochondrial chaperonin 10 from Rattus norvegicus (rat cpn10), with N-terminal acetylation, has been published. Two syntheses of rat cpn10 were performed, the first using a classical carbodiimide-mediated double coupling protocol (Method A) and the second a more efficient HBTU/HOBT/single coupling procedure (Method B). The latter also involved the application of a capping procedure, using N-(2-chlorobenzyloxycarbonyloxy)succinimide [Z(2-Cl)-OSu]. The crude protein from Method A was purified using a two-step isoelectric focusing/RP-HPLC scheme and found to contain a high proportion of a deletion peptide (less Gln60). Conversely, rat cpn10 from Method B was purified to homogeneity by one-step RP-HPLC, using a reversible lipophilic chromatographic probe. The proportion of biologically active heptameric structure was directly related to the purity of the protein and attained 84% with material from Method B. The addition of Ca/Mg ions, pH 7.2, or a heating/cooling cycle increased the proportion of heptamer for less pure protein. Shorter sequences were found not to fold into heptamers, suggesting that aggregation/folding motifs are located in 1-25 and 77-101 regions of rat cpn10. The heptameric cpn10 (Method B) bound correctly to GroEL from E. coli, demonstrating that N-terminal acetylation is not necessary for its folding and binding to bacterial cpn60.

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Four alkylating bombesin (BN) analogues (two C-terminal and one N-terminal chloromethylketone derivatives, and one chloroethylnitrosourea derivative) were synthesized and tested in Swiss 3T3 fibroblasts for receptor binding and mitogenic activity. Although they bound to the BN receptor with no or very weak mitogenic activity, no one analogue inhibited BN-induced thymidine incorporation in the contemporaneous treatment; only one behaved as a weak receptor antagonist when given 24 h before BN stimulation.

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As a continuation of our program to study the structure-function relationship of the peptide hormone somatostatin, we report the synthesis and biological potencies of a series of cyclic hexapeptide analogs related to somatostatin. The parent peptide of this series was designed by Veber and coworkers, c[-Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11-], and has been reported to be superactive in the inhibition of the release of growth hormone. (The superscript numbers refer to the positions of residues in native somatostatin). The series of analogs has been designed to examine the role of the so-called bridging region, Phe11-Pro6, which has been postulated to be important in maintaining the proper conformation of the biologically active tetrapeptide, Phe-D-Trp-Lys-Thr. We have incorporated peptidomimetics and the retro-inverso modification into the bridging region of the molecule, with the aim of affecting the conformational preferences found in the parent peptide. Results from the biological assay--in vitro inhibition of growth hormone--and the conformational analysis (adjoining paper) of our analogs will provide insight into the relationship between structure and biological activity of somatostatin.

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PHI--a new candidate hormone from porcine intestinal tract-- corresponds to a linear heptacosapeptide amide of remarkable sequence homology to the known members of the glucagon family, particularly to the vasoactive intestinal peptide (VIP) and secretin. The position 24 usually occupied by an aminodicarboxylic acid omega-amide, in the present case, however, carries a glutamic acid, thus opening the question of whether this structural feature is related to desamidation in one of the isolation and characterization steps or of whether it is significant for this peptide factor. Consequently the heptacosapeptide amides corresponding to the proposed primary structure and to its 24-glutamine analogue have been synthesized. Comparative chromatographic and biological studies on the natural and the two synthetic products have confirmed the correctness of the primary structure proposed for the isolated PHI. Since [24-glutamic acid] and [24-glutamine]PHI exhibit no significant differences in their biological potencies, the main question is still open of whether the position 24 in native PHI is occupied by the aminodicarboxylic acid omega-amide (glutamine) or by N-substituted derivatives (N-glycosyl).

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