|Siviter, Richard - UNIV OF LEEDS, UK|
|Dani, M - UNIV OF LEEDS, UK|
|Keen, Jeffrey - UNIV OF LEEDS, UK|
|Shirras, Alan - UNIV OF LANCASTER, UK|
|Isaac, R - UNIV OF LEEDS, UK|
Submitted to: Peptides
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 2, 2002
Publication Date: October 18, 2002
Citation: Siviter, R.J., Nachman, R.J., Dani, M.P., Keen, J.N., Shirras, A.D., Isaac, R.E. 2002. Peptidyl dipeptidases (ANCE and ACER) of Drosophila melanogaster: Major differences in the substrate specificity of two homologs of human angiotensin I-converting enzyme. Peptides. 23:2025-2034. Interpretive Summary: Neuropeptides are short chains of amino acids (the building blocks of proteins) that regulate aspects of reproduction, development and digestion that are critical for insect survival. Nevertheless, these insect peptides in and of themselves hold little promise as insect control agents because of susceptibility to being degraded in the target insect, and inability to pass through the outside skin (cuticle) and/or digestive tract of the insect. We must design neuropeptide mimics that resist degradation by enzymes in the digestive tract and blood of pest insects and enter efficiently via a topical or oral route. We report on the comparative ability of two related enzymes that exist in the blood and tissues of flies and other pest insects to degrade a number of different natural neuropeptides. We report as well on various natural conditions that can modify the efficiency of neuropeptide degradation by the two enzymes. This information will aid in the development of enzyme-resistant mimics capable of disrupting neuropeptide-regulated processes in insects. The work brings us one step closer to the development of practical neuropeptide-like substances that will be effective in controlling pest insects in an environmentally friendly fashion.
Technical Abstract: Drosophila melanogaster angiotensin converting enzyme (Ance) and angiotensin converting enzyme related (Acer) are single domain homologs of mammalian peptidyl dipeptidase A (angiotensin I-converting enzyme) whose physiological substrates have not as yet been identified. We have investigated the in vitro susbstrate specificities of the two peptidases towards a variety of insect and mammalian peptides. Ance was generally much better than Acer at hydrolyzing peptides of 5-13 amino acids in length. Only two of the peptides, [Leu5]enkephalinamide and leucokinin-I were cleaved faster by Acer. Increasing NaCl concentration had opposite affects on the cleavage of [Leu5]enkephalin and [Leu5]enkephalinamide by Acer, decreasing the activity towards [Leu5]enkephalin but increasing the activity towards [Leu5]enkephalinamide. Of the insect peptides tested, the tachykinin-related peptide, Lom TK-1, proved to be the best substrate for Ance with kcat/km ratio of 0.122 s-1uM-1. However, in comparison, the D. melanogaster tachykinins, DTK-1, DTK-2, and DTK-3 and DTK-4 were poor Ance substrates. DTK-5 was the best substrate of this family, but the apparent high km for hydrolysis by Ance suggested that this peptide would not be a natural Ance substrate. This low affinity for DTK-5 is the likely reason why the peptide was not rapidly degraded in D. melanogaster hemolymph, where Ance was shown to be a major peptide-degrading activity.