Alain Morel

The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28 (1–12)

The products generated after addition of the ARG-LYS esteropeptidase activity purified from rat brain to synthetic somatostatin-28 were analyzed using radioimmunoassay, HPLC and amino acid analysis. In addition to somatostatin-14, both free arginine and free Lysine were identified together with somatostatin-28. The dipeptide ARG-LYS was not present, which indicates that three peptide bonds were hydrolyzed in order to achieve excision of the doublet. Since it is likely that the octacosapeptide is a precursor for both somatostatin-14 and somatostatin-28, these observations add further support to the hypothesis that the convertase is also involved in the in vivo processing of endogenous somatostatin-28.

Regional distribution of the Mr 15,000 somatostatin precursor, somatostatin-28 and somatostatin-14 in the rat brain suggests a differential intracellular processing of the high molecular weight species

Three different forms of immunoreactive somatostatin (Mr 15,000, 3,000 and 1,600) were immunologically detected in extracts made from six neural structures of the rat brain. The largest represents the proform, while the smaller were identified by high pressure liquid chromatography with bovine somatostatin-28 (S-28) and -14 (S-14) respectively. In each of the brain structures studied highly variable proportions of precursor, S-28 and S-14 were found. These observations provide suggestive evidence that the intracellular processing of the 15,000 Mr proform may occur differently in the various somatostatinergic pathways of the brain. They argue in favor of a biological role for the precursor in providing distinct relative proportions of S-14 and S-28 in specific rat brain regions.

Characterization of a somatostatin-28 generating metallo-endoprotease from rat brain cytosol.

Brain cytosol contains a neutral metallo-protease of about 80,000 which cleaves a substrate containing the site at which mammalian prosomatostatin is cleaved to generate somatostatin 28 in vivo. This represents a cleavage on the carboxyl side of a single arginine residue at an Arg-Ser bond. The enzyme was unable to cleave several other substrates containing single arginine residues or two substrates containing an Arg-Lys or Lys-Arg pair. When it was incubated with anglerfish pancreatic prosomatostatin, it produced significant quantities of a peptide which co-eluted with somatostatin 28 II. Based on the ability of this enzyme to cleave small and large substrates related to somatostatin, it is a potential candidate for the enzymes which cleaves prosomatostatin in vivo.

Enzymes Processing Somatostatin-Precursors, The Somatostatin-28 Convertase of Rat Brain, A System Converting Somatostatin-28 to Both Somatostatins-14 and -28(1–12)

In the biosynthetic pathways of neuropeptides both co- and post-translational events are essential in confering to these messengers their biological activity. Regulation of these mechanisms may be critical in giving rise to diversity in the products derived from the processing of a single, but multipotential, biosynthetic precursor (Eipper and Mains, 1980; Benoit et al., 1982a; Kimura et al., 1986). Of particular importance is the proteolysis which allows the active fragments to be released from their larger proforms. Cleavage of peptide bonds appears to occurs generally at loci consisting in basic amino acids (Arg or Lys) arranged within the precursor sequence as doublets (Nakanishi et al., 1979), sometime as triplets (Craig et al., 1982) or even as quadruplets (Nakanishi et al., 1979; Furutani et al., 1983). Attempts to characterize the enzyme(s) possibly involved in these cleavages were hampered by difficulties in obtaining sufficient amounts of purified hormone precursors. The biosynthetic system of somatostatin-14 (S-14), a tetradecapeptide found both in the central nervous system and in the gastro-intestinal tract, is particularly well-suited for such studies since, in mammals, the peptide hormone seems to derive from a single precursor (Morel et al., 1983). Its structure was determined in various systems through the sequencing of cDNA, to the corresponding mRNAs (Hobart et al., 1980; Goodman et al., 1983). In all cases, the S-14 molecule occupies the COOH-terminal end of the precursor and is preceded at positions -1 and -2 by a basic doublet (Lys-1, Arg-2). Amino terminal extension of this particular structure leads to an octaeicosapeptide, somatostatin-28 (S-28), which includes in its sequence both the NH2-terminal fragment, called S-28 (1–12) and S-14. Therefore, in theory, selective excision of the Arg Lys doublet should release stoichiometric amounts of both S-14 and S-28 (1–12). The corollary is that the octaeicosapeptide S-28 was considered as a possible common precursor to both the NH2- and COOH- terminal dodeca- and tetradeca- peptides respectively (see for a discussion Patel Y.C., 1986).