A search for vertebrate peptides containing D-amino acids

In multicellular organisms many vital functions are mediated by peptides. Some of these peptides contain a D- amino acid in a well-defined position. To date, in all vertebrates and several invertebrates the D-amino acid is the second residue of the mature peptide whereas it has been found in other positions in invertebrates. Vertebrate peptides comprise dermorphins, deltorphins, and the peptide antibiotics bombinin H from frog skin, as well as a C- type natriuretic peptide (CNP) and beta-defensin-like peptide (DLP) from the venom of male Platypus, a primitive mammal. In case of the opioid peptides, the all L-forms are inactive, whereas subtle differences in activity and physicochemical properties between the two diastereomers of bombinin H have been observed. From skin secretions of Bombinae, an enzyme has been isolated which catalyses the posttranslational inversion of the stereochemistry of the respective L-amino acids present in the precursor of bombinin H. This L/D-isomerase acts exclusively on the second residue. Genes encoding polypeptides related to the frog skin enzyme are present in many vertebrates including man supporting the idea of the existence of diastereomeric peptides in higher mammals. A detailed study of the substrate specificity of the frog isomerase and subsequent data base search has led to the identification of potential substrates, which include hormones, antibacterial peptides etc., for which a D-form could exist as well. Based on this knowledge, the proposed project mainly will focus on the questions, whether i) the diastereomeric form of a few selected peptides can be detected in animal tissues and thus the presence of the D- residue be directly confirmed, and ii) the biological activity and/or physicochemical properties of the peptides are modulated by the stereoinversion. In particular, the D-amino acid could crucially affect receptor interactions, folding propensities or polypeptide turnover in these peptides and thus have far-ranging consequences on their biological function.

It is a well-known fact that the molecules of life are mostly homochiral, which means that from two possible chiral (image or its mirror image) molecular structures only one exists in nature. Peptides and proteins, for example, consist of L-amino acids. However, a few exceptions are known so far, where this rule is broken and D-amino acids are incorporated at defined positions. But those may only represent the proverbial tip of the iceberg, as the analytics of D-amino acids is tricky for a number of technical reasons and the routine methods usually employed in peptidomics and proteomics do not detect the presence of a D-residue in a polypeptide. For example, in mass spectrometric (MS) analysis, the retrieved masses are the same for all stereoisomers. There are, however, subtle differences between their spectra. Based on a fingerprint analysis, the chirality can thus be determined in short time and potentially high throughput. By identifying the [D-Leu2] isomer of the predicted peptide antibiotic GH-2 in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MS measurements. In the future, L/D analytics might be possible as a simple computational extension of routine MS analysis of peptides.Furthermore, we investigated two peptide antibiotics, which potentially could contain a D-amino acid at position two in nature. The first was an unusual peptide, aDrs, from the skin secretions of the frog Pachymedusa dacnicolor. Previously, aDrs was found to self-assemble to potentially functional nano-fibrils termed amyloid. Here we found that a chiral switch exists, which determines the 3D arrangement of the fibrils to superstructures. The second peptide to be investigated to greater detail was LL-37, which, together with a few other peptide antibiotics, represents the first line of defence against bacteria and other microorganisms in the human body. Most interestingly, we observed a self-organisation, which apparently resembles that of aDrs with respect to regulation and the presence of a superstructural chiral switch. This amyloid could play a functional role in immune defence in a manner not covered by current models for the mode of action of peptide antibiotics. These results will contribute to the development of new antibiotics, which better act in concert with the body's own defences, but might also indicate a direct functional link to the pathogenesis of amyloidoses or certain neurodegenerative diseases, such as Parkinsons or Alzheimers disease.