Differential processing of hormone precursor. Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells.

Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins-14 and -28 [(1989) EMBO J. 8, 2911-2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins-14 and -28, respectively. Radioimmunochemical analysis of the somatostatin-related products indicated that replacement of either Arg-2-Lys-1 by Asn-2-Asn-1 or of Arg-15 by Asn-15 resulted in the exclusive production of either somatostatin-28 or -14, respectively. Moreover only prosomatostatin[1-76] was detected and no somatostatin-28[1-12] could be measured in cell extracts. Selective suppression of either somatostatin-14 or somatostatin-28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin-14 is exclusively generated by dibasic cleavage at the Arg-2-Lys-1 site of the intact precursor with concomitant production of prosomatostatin[1-76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.     

Prosomatostatin is processed in the Golgi apparatus of rat neural cells.

Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.     

The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites

Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.     

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

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.     

Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder

The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I.     

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.     

Characterization of an endoprotease from rat small intestinal mucosal secretory granules which generates somatostatin-28 from prosomatostatin by cleavage after a single arginine residue

We have extracted, characterized, and partially purified an enzyme from secretory granules from rat small intestinal mucosa which cleaves a synthetic prosomatostatin substrate on the carboxyl side of a single arginine residue. This substrate Leu-Gln-Arg-Ser-Ala-Asn-Ser-NH2 contains the monobasic site at which mammalian prosomatostatin is cleaved in vivo to generate somatostatin-28. This activity was released from the granules by osmotic shock followed by extraction with 500 mM KCl. The enzyme had a molecular weight of about 55,000, a pH optimum of about 7.5, and a Km for the synthetic substrate of 20 microM. It was partially inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, iodoacetate, soybean trypsin inhibitor, and EDTA. It was also very sensitive to aprotinin (complete inhibition at 25 micrograms/ml) but was not inhibited by bestatin, pepstatin, or p-chloromercuribenzoate. This endoprotease was unable to cleave three small trypsin and kallikrein substrates (N alpha-benzoyl-L-arginine ethyl ester, N alpha-benzoyl-DL-arginine p-nitroanilide, and N alpha-benzoyl-L-arginine 7-amido-4-methylcoumarin). It was unable to cleave either the Arg-Asp bond in CCK 12 or the Arg-Glu and Arg-Met bonds of synthetic peptides corresponding to sequences of anglerfish prosomatostatin II situated upstream from the somatostatin-28 domain. These observations together suggest that adjacent amino acids play a role in determining the conformational specificity of the monobasic cleavage. This soluble enzyme was also able to cleave three synthetic substrates containing dibasic residues (Arg-Lys or Lys-Arg) on the carboxyl side of the arginine, although it did so less rapidly than at the monobasic cleavage sites. When incubated with partially purified prosomatostatin from anglerfish pancreas, significant quantities of somatostatin-28 II were produced. All these cleavages were completely blocked by preincubation with aprotinin. Although further work is required to clarify the physiological role of this enzyme, it appears, in view of its catalytic properties, this endoprotease could be involved in the conversion of prosomatostatin to somatostatin-28 in intestine mucosal secretory cells.     

Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites.

The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin-14, there is also a fourteen amino acid N-terminally extended form of the tetradecapeptide, somatostatin-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin-14 or -28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin-I and -II) give rise to somatostatin-14 and -28, respectively. Whereas anglerfish prosomatostatin-II was previously shown to release exclusively somatostatin-28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin-I precursor to somatostatin-28 and -14 as well as to a lysine-extended form of somatostatin-14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin-14 and is involved either directly or indirectly in the release of mature somatostatin-14. The isolation of yeast mutants defective in somatostatin-28 expression (sex mutant) allowed the cloning of a non-essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin-28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono- and dibasic processing sites in yeast.     

Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells

We have previously found that preprosomatostatin is processed accurately to both somatostatin-14 and somatostatin-28 in pituitary gonadotrophs of transgenic mice. The foreign somatostatin peptides have been shown to enter the regulated secretory pathway of these cells. To determine whether accurate preprosomatostatin processing can occur in any neuroendocrine cell, we introduced preprosomatostatin cDNA expression vectors into several different neuroendocrine cell lines. We found that prosomatostatin was cleaved efficiently to somatostatin-14 and somatostatin-28 in RIN 5F and AtT20 cells, but not in GH4 or PC12 cells. The ability of a particular cell type to process prosomatostatin did not correlate with cellular storage capacity and was independent of the level of biosynthesis of the precursor. These data suggest that prosomatostatin processing requires specific pathways which are present in some neuroendocrine cells, but not in others.     

Characterization of three peptides derived from prosomatostatin [prosomatostatin-(1-63)-, -(65-76)- and -(79-92)-peptides] in a human pancreatic tumour.

By using only reverse-phase h.p.l.c., three fragments of prosomatostatin were isolated from an extract of a human pancreatic neuroendocrine tumour that produced somatostatin, vasoactive intestinal polypeptide and gastrin-releasing peptide. The amino acid composition of the peptides indicated that they represented prosomatostatin-(1-63)-peptide, prosomatostain-(65-76)-peptide and prosomatostatin-(79-92)-peptide (somatostatin-14). The identity of prosomatostatin-(1-63)-peptide was confirmed by characterization of the products of digestion with Armillaria mellea (honey fungus) proteinase. Partial micro-sequencing of prosomatostatin-(1-63)-peptide showed that the Gly24-Ala25 bond of preprosomatostatin was the site of cleavage of the signal peptide. Thus human prosomatostatin is a protein of 92 amino acid residues that is proteolytically cleaved in a pancreatic tumour at the site of a dibasic-residue (arginine-lysine) processing site and at a single-monobasic-residue (arginine) processing site.     

In vivo synthesis and processing of rat hypothalamic prosomatostatin

The in vivo incorporation of [3H]phenylalanine into an apparent 15 kDa prosomatostatin was observed in the hypothalamus of rats injected with the labeled amino acid in the third ventricle. Precursor-product relationships were established between this newly synthesized material and both somatostatin-28 and -14.     

Direct evidence for two distinct prosomatostatin converting enzymes. Detection using a rapid, sensitive, and specific assay for propeptide converting enzymes

Many bioactive peptides are initially synthesized via larger precursors from which they are released by proteolytic cleavage at basic amino acids. Some precursors contain more than one final product peptide, multiple copies of a single peptide, or both. Different product peptides can be produced from a common precursor in different tissues. It is not currently known whether this cell-type specific production of bioactive peptides is mediated by different, specific propeptide converting enzymes (PCEs) or by a small number of similar PCEs. To resolve this issue for the conversion of prosomatostatin, the processing of prosomatostatin-I (aPSS-I) and prosomatostatin-II (aPSS-II) to either somatostatin-14 (SS-14) or somatostatin-28 (aSS-28), respectively, was examined in anglerfish islets. Two distinct forms of PSS PCE activity were detected using a rapid, sensitive, and specific assay. Examination of the specificity of these two enzyme activities showed that one proteolytic activity performs the aPSS-I to SS-14 conversion, while the other protease liberates aSS-28 from aPSS-II. The SS-14-generating PCE also cleaves aPSS-II to produce [Tyr7,Gly10]SS-14 (a tetra-decapeptide analog of SS-14) and converts proinsulin to insulin. The aSS-28-generating PCE does not process proinsulin. These results provide direct evidence that different, specific PCEs are required for liberation of SS-14 and aSS-28 from their precursors.     

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.     

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.     

Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Presence of somatostatin-28 like immunoreactivity in the human pancreas

Specific antisera against somatostatin-28 were prepared by absorption of somatostatin-28 antisera with sepharose 4B-somatostatin-14. Indirect immunofluorescence techniques using somatostatin-14 antisera and specific antisera against somatostatin-28 were carried out to elucidate the time of occurrence of somatostatin-28 in the fetal pancreatic islets and to ascertain whether somatostatin-28 was present in the adult pancreatic islets or not, and further to examine whether cells reacting with specific antisera against somatostatin-28 are identical to those reacting with somatostatin-14 antisera or not. Somatostatin-28 like immunoreactivity occurred in the fetal pancreatic islets at 11th week's gestation and was found in all fetal pancreatic islets examined in the present study. It was also found in the adult pancreatic islets. Furthermore, cells reacting with specific antisera against somatostatin-28 in the fetal and adult pancreatic islets were identical to those reacting with somatostatin-14 antisera. Thus, the present study elucidated the presence of somatostatin-28 like immunoreactivity in the human pancreas. However, it could not be decided whether cells reacting with somatostatin-28 antisera contain either only somatostatin-28 or both somatostatin-28 and somatostatin-14; in other words, whether somatostatin-14 is produced from somatostatin-28 or not, since somatostatin-14 antisera had a cross-reactivity to both somatostatin-14 and somatostatin-28.     

Patients with Alzheimer's disease show an increased content of 15 Kdalton somatostatin precursor and a lowered level of tetradecapeptide in their cerebrospinal fluid

The relative proportions of both somatostatin-14 and its precursors somatostatin-28 and the 15 Kdalton prosomatostatin were evaluated by radioimmunoassay in the cerebrospinal fluid of patients with Alzheimer's disease. It was observed that the patients have a lowered content in the tetradecapeptide somatostatin while they exhibit a significant increase in unprocessed 15 Kda precursor. These results indicate that these patients possess impaired processing mechanisms which may be responsible for the lowered content in mature somatostatin-14. These observations may provide a valuable test for the ante-mortem diagnosis of the disease. They are discussed in connection with others suggesting that Alzheimer's patients may be selectively altered in their somatostatinergic neurones of their cerebral cortex (Morrison et al. (1985) Nature 314, 90-92. Roberts et al. (1985) Nature 314, 92-94).     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Reduced somatostatin-28-(1-12)-like immunoreactivity in cerebral cortex of dogs with an Eck fistula and somatostatin molecular forms in brain

Somatostatin-28-(1-12)-like immunoreactivity (S28(1-12)LI) in brains of Eck fistula dogs, prepared as an experimental model of hepatic encephalopathy, was measured. Significant reductions of S28(1-12)LI were observed in all cortical regions of Eck fistula dogs. The reductions of S28(1-12)LI were significantly correlated with decreases in somatostatin-14-like immunoreactivity (S14LI) in the cortical region. The ratios of S28(1-12)LI to S14LI in all cortical regions were not different between Eck fistula and normal dogs. Additionally, no difference in gel chromatographic profiles of S28(1-12)LI and S14LI was observed between Eck fistula and normal dogs. These results imply that reduced somatostatin immunoreactivity in hepatic encephalopathy may be caused not by altered degradation but by reduced production of prosomatostatin. Our S28(1-12)LI assay system could detect prosomatostatin(1-76) and S28(1-12) and the S14LI system prosomatostatin, S28 and S14. S28(1-12)LI/S14LI ratios in cortex were 0.64-0.83 and these were significantly different from those (1.02-1.36) in thalamus, midbrain and medulla. Relative proportions of prosomatostatin (20%) and S28 (23-24%) in cortex were larger than those (6-7% and 5-7%, respectively) in thalamus, midbrain and medulla. The differential distribution of these molecular forms suggests that processing of prosomatostatin in cortex may be different from that in thalamus, midbrain and medulla.