From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascarissuum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 pM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor. With few exceptions, these were rather small molecules containing less than 30 amino acids. As for their biological function, two main groups can be discerned. One comprises a multitude of peptides interacting with specific receptors present in the nervous system, the gastrointestinal tract and other organs of mammals. Many of these peptides are in fact similar or even identical to hormones and neurotransmitters. The second group encompasses antimicrobial peptides, at least some of which exert their function through direct binding to the phospholipid bilayer of cell membranes.Recently, a new family of peptides, the xenoxins, was isolated and characterized from skin secretions of Xenopus laevis (Kolbe et al., 1993). These 66-amino-acid peptides are related to neurotoxins and cytotoxins from snake venoms, but they are apparently devoid of toxic activity. In the course of our analysis of the skin secretion of Bombina bombina, a main fraction containing peptides with an apparent molecular mass of 8-10 kDa was isolated. In view of the earlier findings on xenoxins, we Reprint requests to: G. Kreil, Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria; e-mail: gkreil@oeaw.ac.at.wanted to test whether similar constituents were also present in skin secretion of this species. Here we present our analysis of one of the two main components of this fraction. As we have shown, this is a trypsin inhibitor related to the protease inhibitors from Ascaris (Grasberger et al., 1994; Huang et al., 1994).The peptide has been termed Bombina skin trypsin inhibitor, abbreviated BSTI. Results Isolation and amino acid sequence of BSTISkin secretions from B. bombina were collected and extracted with n-butanol. The aqueous phase was chromatographed over ConA-Sepharose to remove glycoproteins, proteoglycans, mucins etc. The eluate was further fractionated by molecular sieve chromatography (see the Materials and methods). One of the main peaks contained polypeptides with an apparent molecular weight of 8-10 kDa, as shown by SDS-PAGE. Separation of this fraction by reverse-phase HPLC yielded three peaks (see Fig. 1). Of these, the main constituent was investigated f...

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The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations.

The pharmacological modulation of collagen turnover is a strategy potentially useful in different skin conditions. The serine protease inhibitor Serpin A1 and portions of its C-terminal region have been investigated as collagen modulators. To clarify the mechanisms by which the C-terminal 409-418 peptide SA1-III increases extracellular type I collagen levels, to compare its activities range with that of the originator molecule Serpin A1, and to evaluate its efficacy in primary cultures from adult and aged human subjects. The different forms of type I collagen were analyzed by means of western blot in cell lysates and cell-conditioned media of primary human dermal fibroblasts obtained from subjects of different ages. Gelatin zymography was used to investigate the degrading enzymes. Cell viability and in vitro wound healing tests were used to evaluate cell proliferation. The SA1-III peptide increased extracellular collagen levels by reducing degradation, with no effect on cellular biosynthesis or cell proliferation mechanisms. A reduced level of MMP-2 and MMP-9 was also found in cell media upon peptide treatment. No peptide effect was detected on inflammatory mediators gene expression in resting and LPS-stimulated fibroblasts, or in the wound healing test. The SA1-III peptide is a good collagen modulator candidate, protecting collagen against degradation without detectable actions on biosynthesis, acting at reasonably low concentrations, and non-interfering with cell proliferation. It is effective in primary fibroblasts from young and aged subjects. These effects can prove useful in pathological and physiological skin conditions in which collagen degradation is excessive compared to the synthetic capacity.

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients' sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients' sera before gluten-free diet. The peptide II, containing Ac-tTG(553-564)-NH2 sequence cross-linked with deamidated Ac-α2-Glia(63-71)-NH2, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553-564)-NH2 and Ac-α2-Glia(63-71)-NH2 and the corresponding cross-linked peptide II were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments.