Cloning and characterization of Aplysia neutral endopeptidase, a metallo-endopeptidase involved in the extracellular metabolism of neuropeptides in Aplysia californica

Cell surface metallo-endopeptidases play important roles in cell communicat ion by controlling the levels of bioactive peptides around peptide receptor s. To understand the relative relevance of these enzymes in the CNS, we cha racterized a metalloendopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and phy siological levels. The membrane-bound activity cleaved Leu-enkephalin at th e Gly(3)-Phe(4) bond with an inhibitor profile similar to that of the mamma lian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the A plysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific ant i-Aplysia NEP (apNEP) antibodies and by the [I-125]-labeled NEP-specific in hibitor RB104, demonstrating that the apNEP gene codes for the RB104-bindin g protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is pr esent both in the cell bodies and in neurites that travel along the neuropi l and peripheral nerves. When incubated in the presence of a specific NEP i nhibitor many neurons of the buccal ganglion showed a greatly prolonged phy siological response to stimulation, suggesting that NEP-like metallo-endope ptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the s mall cardioactive peptide, as suggested by RP-HPLC experiments. More genera lly, the presence of apNEP in the CNS and periphery may indicate that it co uld play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.