Location: Location not imported yet.Title: Digestion of FMRFamide and nematode FMRFamide-like peptides (nematode FLPs) by the soluble fraction from Panagrellus redivivus homogenate) Author
Submitted to: Journal of Helminthology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/22/2008
Publication Date: 5/14/2008
Citation: Masler, E.P. 2008. Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus. Journal of Helminthology. 82:279-285. Interpretive Summary: Plant-parasitic nematodes attack all crops of agricultural importance, causing over $10 billion in losses annually to U.S. farmers. One problem facing growers is that environmental concerns will result in the elimination within the next few years of the most extensively used chemical pesticide used to control nematodes in the United States. This loss makes the discovery of environmentally and economically sound replacement control agents critical. One approach to discovering new methods to control nematodes is to identify ways to disrupt their normal biochemical processes by using chemicals that occur naturally inside the nematode. We have discovered that small protein molecules called FLPs, which are produced naturally by nematodes and are essential for their survival, are degraded in specific ways by nematode enzymes called proteases. The discovery is significant because it shows that specific types of proteases are a key to FLP metabolism and can be used to control the nematodes. Consequently, this information will be used by researchers in the agrochemical and agricultural biotechnology industries who are developing safe, selective methods for nematode control.
Technical Abstract: Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolyzed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1), APKPKFIRFa (FLP-5), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12), and KHEYLRFa (FLP-14) in vitro. Results were assessed by analyzing reaction components with RP-HPLC, UV detection at 210nm, and peak integration. Based upon the peak size for substrate and products, more than 95% of most peptide substrates were consumed after 1hr at 270C. However, arrays of product peaks from each substrate revealed varied complexities in digestion patterns, even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (76% loss; P < .0001), while FLP-14 was digested less (84% loss; P < .0004) than all but FLP-14. Examination of digestion patterns of FLP-12, a second nonapeptide (FLP-5), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviors of each sequence to protease cleavage. Amastatin significantly (P < .02) reduced digestion of FLP-12 (55% loss) and FMRFa (64% loss; P < .01), but had no effect on FLP-5. AEBSF had no protective effect on FMRFa or FLP-12 digestion but significantly decreased (78% loss; P < .0001) hydrolysis of FLP-5. The combination of both inhibitors had additive effects only for FMRFa (40% loss; P < .05). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < .0001) from all proteolytic digestion, whereas FMRdFa digestion was the same as FMRFa. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.