Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3.

Previous studies using selectively modified pro-ocytocin/neurophysin substrate analogues and the purified metalloprotease, pro-ocytocin/neurophysin convertase (magnolysin; EC 3.4 24.62), have shown that dibasic cleavage site processing is associated with a prohormone sequence organized in a beta-turn structure. We have used various peptide analogues of the pro-ocytocin-neurophysin processing domain, and recombinant prohormone convertase 1/3, to test the validity of this property towards this member of the family of prohormone convertases (PCs). The enzymatic cleavage analysis and kinetics showed that: (a) with methyl amide (N-Met) modification, a secondary structure beta-turn breaker, the enzyme substrate interaction was abolished; (b) cleavage was favoured when the dibasic substrate side-chains were oriented in opposite directions; (c) the amino acid present at the P'1 position is important in the enzyme-substrate interaction; (d) the flexibility of the peptide substrate is necessary for the interaction; (e) Addition of dimethylsulfoxide to the cleavage assay favoured the cleavage of the pro-ocytocin/neurophysin large substrate over that of the smaller one pGlu-Arg-Thr-Lys-Arg-methyl coumarin amide. These data allowed us to conclude that proteolytic processing of pro-ocytocin-related peptide substrates by PC1/3 as well as by the metalloenzyme, magnolysin, involves selective recognition of precise cleavage site local secondary structure by the processing enzyme. It is hypothesized that this may represent a general property of peptide precursor proteolytic processing systems.

Predominant basolateral proteolytic processing of prosomatostatin into somatostatin-28 in polarized LLC-PK1 cells.

Polarized epithelial cells secrete specific proteins through their apical or basolateral membrane. In the present study, we have expressed the human prosomatostatin cDNA in the pig kidney epithelial cell line (LLC-PK1) and monitored the processing and release of the somatostatin-related peptides. Analysis by high-performance liquid chromatography and radioimmunoassay of the somatostatin-related peptides synthesized by the transfected cells showed that the LLC-PK1 cells released prosomatostatin and somatostatin-28 (S-28) in the culture medium. Furthermore, when the cells were polarized, we observed release of prosomatostatin from both membrane domains (apical and basolateral), while liberation of S-28 was mostly from the basolateral side. This observation suggests that, in these cells, the proprotein convertase(s) responsible for prosomatostatin processing is(are) associated with the basolateral secretory pathway.

Dipeptidyl-peptidase IV secreted by Aspergillus fumigatus, a fungus pathogenic to humans.

A dipeptidyl-peptidase IV was purified from the culture medium of the human-pathogenic fungus Aspergillus fumigatus. The enzyme has an apparent molecular mass of 95 kDa and contained approximately 10 kDa of N-linked carbohydrate. This glycoprotein is antigenic and has all characteristics of the class IV dipeptidyl-peptidases: removal of Xaa-Pro and to a lesser extent Xaa-Ala dipeptides from the N termini of peptides, including bioactive peptides such as neuropeptide Y, [des-Arg1] bradykinin, and glucagon-like peptide 1, activity at neutral pH, and presence in the amino acid sequence of the Gly-X-Ser-X-Gly consensus motif of the serine-hydrolases and the putative catalytic triad (Ser613, Asp690, His725) of the dipeptidyl-peptidases. Moreover, the last 200 amino acids displayed 60 to 65% similarity with the other dipeptidyl-peptidases IV from rat, mouse, human, and yeast. However, unlike the other dipeptidyl-peptidases, the dipeptidyl-peptidase IV of A. fumigatus is a secreted enzyme with a cleavable signal peptide. Expression of a recombinant dipeptidyl-peptidase IV of A. fumigatus has been attained in the yeast Pichia pastoris.

Role of prohormone convertases in pro-neuropeptide Y processing: coexpression and in vitro kinetic investigations.

Proneuropeptide Y (ProNPY) undergoes cleavage at a single dibasic site Lys38-Arg39 resulting in the formation of 1-39 amino acid NPY which is further processed successively by carboxypeptidase-like and peptidylglycine alpha-amidating monooxygenase enzymes. To investigate whether prohormone convertases are involved in ProNPY processing, a vaccinia virus derived expression system was used to coexpress recombinant ProNPY with each of the prohormone convertases PC1/3, PC2, furin, and PACE4 in Neuro2A and NIH 3T3 cell lines as regulated neuroendocrine and constitutive prototype cell lines, respectively. The analysis of processed products shows that only PC1/3 generates NPY in NIH 3T3 cells while both PC1/3 and PC2 are able to generate NPY in Neuro2A cells. The convertases furin and PACE4 are unable to process ProNPY in either cell line. Moreover, comparative in vitro cleavage of recombinant NPY precursor by the enzymes PC1/3, PC2 and furin shows that only PC1/3 and PC2 are involved in specific cleavage of the dibasic site. Kinetic studies demonstrate that PC1/3 cleaves ProNPY more efficiently than PC2. The main difference between the cleavage efficiency is observed in the Vmax values whereas no major difference is observed in Km values. In addition the cleavage by PC1/3 and PC2 of two peptides reproducing the dibasic cleavage site with different amino acid sequence lengths namely (20-49)-ProNPY and (28-43)-ProNPY was studied. These shortened ProNPY substrates, when recognized by the enzymes, are more efficiently cleaved than ProNPY itself. The shortest peptide is not cleaved by PC2 while it is by PC1/3. On the basis of these observations it is proposed, first, that the constitutive secreted NPY does not result from the cleavage carried out by ubiquitously expressed enzymes furin and PACE4; second, that PC1/3 and PC2 are not equipotent in the cleavage of ProNPY; and third, substrate peptide length might discriminate PC1/3 and PC2 processing activity.

Structural investigation and kinetic characterization of potential cleavage sites of HIV GP160 by human furin and PC1.

A key event in the biosynthesis of the human immunodeficiency virus is the maturation of the gp160 precursor generating gp120 and gp41, two proteins that are fundamental for the infective process. In vivo, gp160 is specifically cleaved at the 515-519 site (REKR decreases A), in spite of the presence in its sequence of another consensus sequence KAKR decreases R (residues 507-511). Comparative kinetic studies on synthetic peptides reproducing different sequences of gp160 by the enzymes PC1 and furin are reported in this paper. The data demonstrate the higher efficiency of furin in the cleavage of peptidic substrates with respect to PC1 and its preference for REKR decreases A vs. KAKR decreases R. Furthermore, furin and PC1 are unable to process peptides patterned on the sequence 307-330 of specific viral strains of the gp120 V3 loop.

Comparative proteolytic processing of rat prosomatostatin by the convertases PC1, PC2, furin, PACE4 and PC5 in constitutive and regulated secretory pathways.

Recombinant vaccinia virus vectors were used to coexpress each of the candidate prohormone convertases PC1, PC2, furin, PACE4 and PC5 with rat prosomatostatin (rProSOM) in the constitutive secreting cell line LoVo and in the endocrine corticotroph cell line AtT-20, which exhibits regulated secretion. Mammalian ProSOM is cleaved at a dibasic Arg-Lys decreases site to produce somatostatin-14 (S-14) and at a monobasic Gln-Arg decreases site to yield somatostatin-28 (S-28). The analysis of processed products by gel-permeation high performance liquid chromatography shows that in LoVo cells PC1, furin and PACE4 generate S-14, S-28 and a mixture of S-14 and S-28, respectively, while PC2 is unable to process ProSOM in these constitutive cells. In contrast, PC2 can generate S-14 in AtT-20 cells. The convertase PC5 is unable to process ProSOM in either cell line. These data suggest that PC2, PC1 and PACE4 are candidate S-14 convertases, while PACE4 and furin are candidate S-28 convertases.

Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site.

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded. Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated. But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite. We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.

Processing of human prosomatostatin in AtT-20 cells: S-28 and S-14 are generated in different secretory pathways.

Somatostatin-14 (S-14) and somatostatin-28 (S-28) are generated by differential processing of a single precursor at a dibasic (R-K) or monobasic (R) proteolytic cleavage site, respectively. To study the pathways of processing of prosomatostatin, we have expressed in AtT20 cells cDNA encoding human prosomatostatin and prosomatostatin mutated in one or the other processing site. Analysis of the peptides present in cell extracts or culture media before and after stimulation of the cells with 8-BrcAMP indicated that prosomatostatin can enter three distinct secretory pathways where it is differently processed: 1) prosomatostatin was secreted through the constitutive pathway; 2) the regulated secretory pathway generated S-14 which was released upon stimulation of the cells with 8-BrcAMP; 3) an alternative pathway, insensitive to 8-BrcAMP produced S-28 and S-14. Moreover, our results suggest that the R-K processing site used to produce S-14 is an important structural feature for targeting the precursor to the regulated secretory pathway.

Prosomatostatin processing in Neuro2A cells. Role of beta-turn structure in the vicinity of the Arg-Lys cleavage site.

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) EMBO J. 8, 2911-2916]. 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 beta-turn-promoting sequence Pro-Arg-Glu-Arg, located near the somatostatin-14 cleavage site and predicted to form a beta-turn structure, was replaced by Ser-Ser-Asn-Arg or Tyr-Lys-Gly-Arg, which have been shown by X-ray diffraction to form beta 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-5] prosomatostatin, [Ser-5, Ser-4, Arg-3] prosomatostatin and [Tyr-5, Lys-4, Gly-3] 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 beta turn structure in the corresponding peptide sequences, replacement of Pro-5-->Ala resulted in a dramatic increase in alpha-helix accompanied by the significant decrease of other structures including beta turn. The data support the hypothesis that the proline residue near the processing site for somatostatin-14 production is an important structural feature for conferring on the cleavage domain the adequate conformation for accessibility to processing enzymes and permitting production of equivalent amounts of both hormones.

Role of beta-turn in proteolytic processing of peptide hormone precursors at dibasic sites.

Proteolytic activation of prohormones and proproteins occurs most frequently at the level of basic amino acids arranged in doublets. Previous predictions by Rholam et al. [Rholam, M., Nicolas, P., & Cohen, P. (1986) FEBS Lett. 207. 1-6] have indicated, on the basis of 20 prohormone sequences containing 53 dibasic potential processing sites, that dibasic sites situated in, or next to, beta-turns were cleaved in vivo, whereas sites included in ordered structures like beta-sheets or alpha-helices were not. We have used peptide analogs of the proocytocin/neurophysin processing domain and a purified preparation of the putative proocytocin convertase from bovine tissues as a model to demonstrate that (1) processing at dibasic sites is associated with a prohormone sequence organized in a beta-turn structure; (2) the beta-turn is an interchangeable motif since the original sequence could be replaced by an heterologous one possessing the ability to organize as a beta-turn; and (3) this particular secondary structure participates in the catalytic reaction, most likely by favoring the interactions of the substrate with the processing endoprotease. It is concluded that, in addition to the dibasic and other amino acids around the cleavage loci, the beta-turn constitutes a key feature in the proteolytic processing reaction in participating as the favorable conformation for optimal substrate-enzyme active site recognition.

Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: the pro-ocytocin model.

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the proforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam, M., Nicolas, P. and Cohen, P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. beta-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a beta-turn in solution; (ii) the sequential organization of the residues participating in the beta-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Synthesis and processing of pro-ocytocin in bovine corpus luteum and granulosa cells.

Bovine corpus luteum is the site of intense production of pro-ocytocin-neurophysin mRNA at day 1 after estrus (Ivell et al. (1985) FEBS Lett. 190, 263-267) which is followed by apparent delayed production of ocytocin. Therefore it is a good model to study both the translational and post-translational production of this neuropeptide in non-hypothalamic tissues and its regulation. In order to assess if this mRNA is translated during the lag period we have analyzed the neurophysin-like species produced in this organ. As early as day 2 after estrus one neurophysin species (pI approximately 4.7) could be detected and was unequivocally identified as pro-ocytocin-neurophysin. In primary cultures of luteinizing granulosa cells, biosynthetic intermediates were characterized, i.e. ocytocin-Gly, ocytocin-Gly-Lys and ocytocin-Gly-Lys-Arg, whereas amidated, fully mature, ocytocin was undetectable. We conclude that translation of pro-ocytocin-neurophysin mRNA takes place soon after transcription and we propose that incomplete processing could be responsible for the low level of ocytocin in the early bovine corpus luteum.

Evidence for beta-turn structure in model peptides reproducing pro-ocytocin/neurophysin proteolytic processing site.

The structural organization of small peptides reproducing the amino acid sequence of the common ocytocin/neurophysin precursor around the LysArg cleavage locus was investigated by a combination of spectroscopical techniques. In water both circular dichroism and [1H] NMR spectra indicated that these peptides adopted a random conformation. Evidence for folded structures was obtained when these compounds were placed in a membrane-like environment i.e. 40 mM SDS in phosphate buffer or trifluoroethanol. Whereas the CD spectra indicated the formation of various types of beta-turn in rapid equilibrium, measurements of NH temperature coefficients and Nuclear Overhauser Effects by 400 and 500 MHz NMR revealed the existence of contacts and of a folded conformation. These observations are discussed in relation with previous hypothesis made on the secondary structure organization of the proteolytic processing site of polypeptide hormone precursors.

Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones. The pro-ocytocin/neurophysin model.

Pro-ocytocin/neurophysin convertase is a divalent cation-dependent endoprotease isolated from both bovine corpus luteum and neurohypophyseal secretory granules. The putative pro-ocytocin/neurophysin converting enzyme cleaves the Arg12-Ala13 bonds of both pro-ocytocin/neurophysin (1----20) and pro-ocytocin/neurophysin obtained by hemisynthesis. The minimal efficient substrate structure allowing recognition by this processing endoprotease was defined by measuring its cleavage efficiency and the inhibitory properties of a set of 34 selectively modified derivatives of the (1----20) NH2-terminal domain of the ocytocin/neurophysin precursor. The data demonstrate that: (i) the basic Lys11-Arg12 doublet, although necessary, is not sufficient; (ii) a minimal substrate length of nine amino acids (residues 7-15 or 8-16) is essential; (iii) those amino acids around the Lys-Arg doublet which contribute to the formation of a possible beta-turn-alpha-helix secondary structure are critical; (iv) substrate recognition by the enzyme may involve several subsites in which structural determinants, situated on both sides of the basic doublet, participate; (v) the NH2-terminal sequence of neurophysin plays a critical role in the correct reading of the cleavage sequence by the processing endoprotease. It is proposed, first, that this type of structural feature may constitute the basis of a general coding system for endoproteases involved in the processing of polypeptide hormone precursors; second, that in addition to its role in the intragranular packaging of the nonapeptide hormone, neurophysin plays a key role in the correct processing of its common precursor with ocytocin.

Proocytocin/neurophysin convertase from bovine neurohypophysis and corpus luteum secretory granules: complete purification, structure-function relationships, and competitive inhibitor.

Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites.