Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine‐ and leucine‐enkephalins. For this reason this enzyme is often called ‘enkephalinase’. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N‐terminal cytoplasmic domain (27 amino acids), a single membrane‐spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn‐metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase.

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His‐142 and His‐146) and a third histidine (His‐231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His‐583, His‐587 and His‐637) was explored by site‐directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS‐1 cells. Substitution of either His‐583 or His‐587 of NEP for Phe completely abolished the activity and Zn‐directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His‐ 142 and His‐146 of thermolysin as zinc ligands. In contrast, substitution of His‐637 for a phenylalanine residue was without effect on enzyme activity.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site‐directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta‐turn structures in the immediate vicinity of the somatostatin‐28 (S‐28) and somatostatin‐14 (S‐14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S‐28 and S‐14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta‐turns had various effects on prosomatostatin processing: substitution of Ala for Pro‐5 drastically decreased prosomatostatin processing and replacement of Pro‐9 by Ala led to the accumulation of the intermediate maturation product [Arg‐2Lys‐1]‐S‐14. In contrast, substitution of Ala for Asn‐12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.

Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14[Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBU J. 8, 2911 -29161. In this study, site-directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular-dichroism and Fourier-transform-infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position -5 and the P-turn-promoting sequence Pro-Arg-Glu-Arg, located near the somatostatin-1 4 cleavage site and predicted to form a p-turn structure, was replaced by Ser-Ser-Asn-Arg or Tyr-Lys-Gly-Arg, which have been shown by X-ray diffraction to form p turns in other proteins. Analysis of the prosomatostatin-derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro-5 + Ala abolished cleavage at the dibasic site, the formation of mutants [Gly-'] prosomatostatin, [Ser-', S e r 4 , Arg-3] prosomatostatin and [ T y P , L y s~~, G~Y -~] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a p turn structure in the corresponding peptide sequences, replacement of Pro-5 + Ala resulted in a dramatic increase in a-helix accompanied by the significant decrease of other structures including fi turn.

Neuro 2A cells infected with a retroviral ' :ctor carryin|t human prosomatostatin eDNA expressed and proces~d con'eetly ihc precursor into soma. tostatins-14 and .28 [(1989} EMBO J, 1~. 2911, in order to stud~, the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (AqlLysl or monobasic [Argl cleavage sites involved in the produclion of somalostatin=. 14 and .28, respectively, Radioimmtmochemie:d amtly~is ~f the somatostafin.rdated product~ indicated tha! repl:lcement of either Arg"-'.Ly~* ~ by AIn ==-Ash" b or of Ar~" ~j' by Asn'~ resulted In the exclusive production ofeither somatostatin.28 or-14. respectively, Moreover only prosomatostatin [ b, 76] was detected and no somatostatin-28[l-12] could b¢ measured in cell extracts, Selective suppression of either somatostatm. 14 or somatostatln.2l~l release by mutation did not affect the I¢v¢1 of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatia, Iris concluded that in this cell type (i) somatostatin.14 is exclusively generated by dibasic cle~val~e at the Arg"a-Lys "~ site of the intact precursor with concomitant productr ~n of prosomatostatin [ I~76], and (ill no direct interactions between the monobasie and dibasic processing domains occur.