Peptide hormones and neuropeptides are initially synthesized as precursor proteins that are subsequently cleaved by limited proteolysis. Aberrations of this process may be responsible for some diseases of idiopathic hormone hypofunction. Although considerable information has accumulated regarding the molecular characterization and action of prohormones and their cleaved peptide products, there is limited information about the molecular characteristics, action, and localization of the enzymes which are responsible for their processing. A major problem has been the isolation of minute amounts of processing enzymes from relatively complex tissues. Research described in this proposal takes advantage of a unique prohormone processing system in Aplysia californica relating to egg-laying hormone (ELH), in which many of the problems identified in characterizing prohormone processing mechanisms in mammalian systems are minimized. ELH is a neuropeptide which elicits egg-laying behavior in Aplysia and is synthesized as a precursor protein by bag cell neurons of the abdominal ganglion. Aplysia also possesses an exocrine organ, the atrial gland, which expresses genes that are related to ELH, and produces peptides homologous to ELH which will induce egg-laying behavior when injected into an animal. Since the atrial gland produces large quantities of ELH-related precursor protein is readily accessible, and its prohormone-like processing mechanism is not complex, it provides a rare opportunity to isolate prohormone-like processing enzymes. The bag cell neurons probably utilize processing enzymes that are identical or similar to those in the atrial gland, since the precursors and peptide products formed are homologous. Processing mechanisms in the bag cell neurons indicate to be of intermediate complexity when compared to mammalian systems, and are likely to involve packaging and routing mechanisms. Research is proposed to isolate the dibasic endoprotease involved in processing ELH-related precursors in the atrial gland, determine its structure, establish its intracellular location, and demonstrate its intragranular activity. These studies will be carried out using biochemical and molecular cloning techniques, Northern hybridization, immunocytochemistry, and in situ hybridization. Monoclonal antibody probes will be developed to identify the cleavage of specific dibasic sites within granules by immunocytochemistry. Development of the model will initiate with the atrial gland and will be expanded to include the bag cell neurons, as well as other neurons expressing ELH-like genes.
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