Neuropeptide amidation: cloning of a bifunctional alpha-amidating enzyme from Aplysia

One of the most common mechanisms of posttranslational modifications to gen erate biologically active (neuro)peptides is the process of peptide alpha - amidation. The only enzyme known to catalyze this important modification is peptidylglycine alpha -amidating monooxygenase (PAM): a (bifunctional) zym ogen, giving rise to a monooxygenase (PHM) and a lyase (PAL). The highly pe ptidergic central nervous system and endocrine system of the marine mollusk Aplysia has homologs of various mammalian peptide processing enzymes, incl uding furin, Afurin2, prohormone convertase 1 (PC1), PC2, carboxypeptidase E (CPE) and CPD. Previously, it has been shown that the abdominal ganglion of Aplysia, which contains similar to 800 peptidergic bag cell neurons, con tains the highest specific alpha -amidating activity. We have identified an d cloned multiple overlapping central nervous system and bag cell cDNAs tha t encode a predicted 748-residue protein that is a member of the PAM family . The protein sequence contains the contiguous sequence of the catalytic do mains of PHM and PAL, clearly demonstrating the existence of bifunctional A plysia PAM, the first invertebrate PAM zymogen with an organization similar to that in vertebrates. None of the characterized clones encoded the so-ca lled exon A domain between the PHM and PAL domains. Furthermore, in a speci fic search by reverse transcription-polymerase chain reaction of RNA from m ultiple tissues we could only detect exon A-less transcripts. PAM expressio n was detected in the central nervous system, and in several endocrine and exocrine organs. Aplysia PAM is a candidate prohormone processing enzyme th at plays an important role in the processing of Aplysia prohormones in the secretory pathway. (C) 2000 Elsevier Science B.V. All rights reserved.