Potential mammalian homologues of a peptidyl-L/D-isomerase

Amphibian skin contains many interesting bioactive peptides. In some of these, the second residue is a D-amino acid. Examples are dermorphins and deltorphions as well as the peptide antibiotics bombinin H. In case of the opioid peptides, the all L-forms are inactive, whereas subtle differences in activity between the two diastereomeres of bombinin H have been reported. As shown earlier by cDNA cloning, the D-amino acids are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, an enzyme has been isolated which catalyses the isomerization of an L-isoleucine in position two of a model peptide to D- allo-isoleucine. This L/D-isomerase acts without any added cofactor via deprotonation-protonation of the alpha- carbon of the second residue. From partial amino acid sequences, cloned cDNA and genomic DNA, the primary structure of this isomerase could be deduced. Genes encoding polypeptides related to the frog skin enzyme are present in many vertebrates including man. Based on these results, the proposed project will focus on two main questions: i) determination of substrate specificity with the aim to get an idea, which other peptides from frogs and possibly other vertebrates potentially could contain a D-amino acid, and ii) do genes related to the frog isomerase present in different vertebrates code for an active L/D-isomerase ? In particular, the expression of part of a human gene coding for the amino-terminal domain of an Fc-gamma binding protein with significant homology to the frog isomerase will be studied: The presence of such an enzyme in certain tissues or secretions raises the possibility that substrates, i.e. peptides where an L-amino acid is converted to the D-isomer, are present as well. These could be known hormones, neurotransmitters, antibacterial peptides etc., for which a diastereomere exists as well.

In the skin of fire-bellied toads (Bombina species), a peptidyl-aminoacyl-L/D-isomerase is present which catalyses the posttranslational isomerization of the L- to the D-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. This isomerase utilizes a deprotonation/protonation mechanism. We have synthesized peptidic substrate analogues with planar alpha,beta-unsaturated residues at the second position. These compounds were found to be potent inhibitors of the enzyme reaction. Our results strongly support the view that the enzyme reaction proceeds via a planar transition state, namely an enolate anion intermediate. Moreover, we analysed the substrate specificity of this isomerase using fluorescence-labelled variants of the natural substrate bombinin H. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. Expression of such an enzyme with a broad specificity in other species would probably result in the formation of peptides containing a D-amino acid. Indeed, numerous peptides from amphibia and mammals are deposited in the data banks, which fulfil the requirements determined here. The question whether such peptides rarely occur in nature or whether we have only seen the proverbial tip of the iceberg remains open, yet the latter becomes more likely. A potential substrate for such an enzyme is anionic dermaseptin (aDrs) from the skin of the Mexican tree frog, which has a negative net charge at neutral pH. In an initial study, we obtained no evidence for antibacterial or cytotoxic activity of soluble aDrs. Conversely, the peptide self-assembles into amyloid fibrils in a reversible, pH- controlled fashion. Interestingly, metastable amorphous aggregates, which are quickly released from an amyloidous depot by a shift in pH, can mediate a strong cytotoxic effect; aDrs can thus be regarded as another candidate for functional amyloid. The bombinins H, the natural substrates of the Bombina L/D-isomerase, are particularly rich in glycine (25 %), which also may allow structural polymorphism and peptide-peptide interactions. Indeed, under conditions, which trigger self-aggregation of these hydrophobic peptides, we found that the stereochemistry of the randomly ordered N-terminal segment modulates the preference to fold into a particular conformation. Therefore, in these members of the emerging class of self-aggregating frog skin peptides, stereochemistry could play an important role in the modulation of self-assembly with far-ranging consequences on the innate immune system, for which amphibian skin is a well-approved model system.