The role of paired basic amino acids in mediating proteolytic cleavage of prosomatostatin. Analysis using site-directed mutagenesis

Many small peptide hormones are synthesized as larger precursors in which the mature hormone sequence is flanked by pairs of basic amino acids. These precursors often undergo extensive post-translational modifications; a critical step in this process is proteolytic excision of the hormone at the paired basic residues. To determine the role of paired basic amino acids as recognition signals for cleavage by processing enzymes, we investigated the heterologous expression of prosomatostatin (the pro-somatotropin release inhibiting factor (pro-SRIF). Pro-SRIF is one of the simplest peptide hormone precursors, possessing a single copy of the 14-residue SRIF peptide at its carboxyl terminus preceded by the least common pair of basic amino acids, Arg-Lys. Employing site-directed mutagenesis, we altered the paired basic cleavage site to the more common Arg-Arg and Lys-Arg residues. The native and mutated precursors were expressed in rat pituitary GH3 cells and mouse 3T3 cells using a retroviral vector. Alteration of the paired basic residues had no effect on the specificity of proteolytic cleavage as both the native and mutant precursors were processed with 70 to 80% efficiency in GH3 cells. Surprisingly, when the mutant pro-SRIFs were expressed in 3T3 cells, which do not process the native precursor, the Arg-Arg and Lys-Arg precursors were processed with 16 and 20% efficiency, respectively. The role of an acidic compartment in mediating pro-SRIF cleavage was also investigated using low concentrations of the lysosomotrophic drug Chloroquine. Twenty-five microM Chlorquine completely inhibited pro-SRIF cleavage and intracellular storage; the unprocessed precursor was secreted into the medium. We conclude that (i) exposure to an acidic compartment is required for pro-SRIF maturation, and (ii) the conformation of the processing site, rather than the composition of the basic amino acids, defines cleavage specificity by prohormone processing enzymes.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Purification and characterization of a paired basic residue-specific prohormone-converting enzyme from bovine pituitary neural lobe secretory vesicles

The neuropeptides arginine vasopressin and oxytocin are generated from their prohormones in the hypothalamoneurohypophysial system by enzymatic cleavages at paired basic residues (i.e. Lys-Arg). This study describes the purification of an enzyme from bovine neural lobe secretory vesicles, the putative site of this processing, which is capable of cleaving several prohormones at paired basic residues. The enzyme is a glycoprotein of Mr approximately 70,000 and has an acidic pH maximum. It processes the heterologous precursors pro-opiomelanocortin and insulin at paired basic residues in a manner similar to a pro-opiomelanocortin-converting enzyme derived from bovine intermediate lobe secretory vesicles which has been described previously. In addition, the neural lobe-derived converting enzyme cleaves the human vasopressin prohormone in vitro to yield arginine vasopressin-Gly10-Lys11-Arg12 as the major vasopressin cleavage product. This indicates that the enzymatic cleavage in the vasopressin precursor occurred primarily on the carboxyl side of the arginine in the pair of Lys-Arg basic residues separating the vasopressin peptide from the neurophysin moiety in the precursor. The properties of the neural and intermediate lobe-derived enzymes are virtually identical, raising the possibility that a family of similar enzymes may be responsible for cleaving a number of prohormones at paired basic residues in different tissues.     

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.     

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.     

Consensus sequence for processing of peptide precursors at monobasic sites

Many regulatory peptide precursors undergo post-translational processing at mono- and/or dibasic residues. Comparison of amino acids around the monobasic cleavage sites suggests that these cleavages follow certain sequence motifs and can be described as the rules that govern monobasic cleavages: (i) a basic amino acid it present at either 3, 5, or 7 amino acids N-terminal to the cleavage site, (ii) hydrophobic aliphatic amino acids (leucine, isoleucine, valine, or methionine) are never present in the position C-terminal to the monobasic amino acid at the cleavage site, (iii) a cysteine is never present in the vicinity of the cleavage site, and (iv) an aromatic amino acid is never present at the position N-terminal to the monobasic amino acid at the cleavage site. In addition to these rules, the monobasic cleavages follow certain tendencies: (i) the amino acid at the cleavage site tends to be predominantly arginine, (ii) the amino acid at the position C-terminal to the cleavage site tends to be serine, alanine or glycine in more than 60% of the cases, (iii) the amino acid at either 3, 5, or 7 position N-terminal to the cleavage site tends to be arginine, (iv) aromatic amino acids are rare at the position C-terminal to the monobasic amino acid at the cleavage site, and (v) aliphatic amino acids tend to be in the two positions N-terminal to and the two positions C-terminal to the cleavage site, except as noted above. When compared with a large number of sequence containing single basic amino acids, these rules and tendencies are capable of not only correctly predicting the processing sites, but also are capable of excluding most of the single basic sequences that are known to be uncleaved. Many or these rules can also be applied to correctly predict the dibasic and multibasic cleavage sites suggesting that the rules and tendencies could govern endoproteolytic processing at the monobasic, dibasic and multibasic sites.     

Roles of divalent metal ions in flap endonuclease-substrate interactions

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.     

Purification and characterization of a putative proenkephalin cleaving enzyme.

A putative proenkephalin-cleaving enzyme (PCE) extracted from bovine adrenal chromaffin granules was purified with soybean trypsin inhibitor high-performance affinity chromatography. The 12,600-fold purified enzyme was maximally active at pH 8.0. The enzyme was completely inhibited with lima bean trypsin inhibitor (0.1 mg/ml), soybean trypsin inhibitor (0.1 mg/ml), and p-(chloromercuri)benzenesulfonic acid (1.0 mM), indicating PCE is a serine protease with cysteine residues likely to be involved in its structure or activity. It exhibited significant autoproteolysis without specific substrates present. The substrate specificity and kinetic constants with the enkephalin-containing (EC) peptides Leu-9 and proenkephalin Peptides B, E, and F as substrates were studied. The cleavage patterns were substantially different than with trypsin digestion. PCE specifically recognized the paired basic amino acid residues and predominantly cleaved the peptide bonds between Lys and Arg sites and peptide bonds after Lys-Lys and Arg-Arg sites. Different Km and Vmax values for the different Lys-Arg sites indicate sequences in addition to the paired basic residues can affect enzyme activity. Also, the lower Km and Vmax of Peptide E suggest a higher affinity for this peptide but much slower cleavage. The C-terminally located Lys-Arg site appears responsible for this high affinity. Based on these observations, we propose the following: (a) the primary structure of these peptides contains enough information to be processed correctly by PCE and (b) PCE may be regulated by pH and Peptide E to prevent extensive processing of the intermediate EC peptides which are the major opioid peptides found in the adrenal chromaffin granules.     

An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes

Most peptide hormones and neurotransmitters are synthesized as larger precursor proteins, which are post-translationally processed to mature bioactive products. An early event in prohormone maturation is endoproteolytic cleavage, occurring usually at pairs of basic amino acids (e.g. Lys-Arg). Since many of the characteristics of a prohormone endoprotease are unknown, distinguishing these enzymes from other cellular proteases in vitro has been difficult. In this report, the substrate specificity of a model prohormone processing system, the insulinoma cell line Rin m5F, was characterized in vivo to establish a set of criteria by which putative proinsulin endoproteases may be assessed. To determine the role of composition of the paired basic amino acid site in directing cleavage, a series of mutant prohormones containing altered cleavage sites was constructed and expressed in Rin m5F cells. Proopiomelanocortin (POMC) was used as a substrate since this prohormone was previously shown to be processed by these cells. To control for positional effects, all four permutations of lysine and arginine (Lys-Arg, Arg-Arg, Arg-Lys, and Lys-Lys) were introduced at both the efficiently processed cleavage site separating the ACTH and beta-lipotropin (beta-LPH) domains of POMC and at the inefficiently processed site in the beta-endorphin sequence near the COOH-terminus of the precursor. His-Arg and Met-Arg sites were also introduced at the ACTH/beta-LPH junction to assess the requirement for paired lysines and arginines. Identification of POMC-derived peptides demonstrated efficient processing of Lys-Arg and inefficient processing of Lys-Lys and Arg-Lys sites at both positions in the prohormone. The Arg-Arg sequence, however, was processed in a position-dependent manner, being efficiently cleaved between ACTH and beta-LPH but only about 50% processed within beta-endorphin. His-Arg was not cleaved in Rin m5F cells, although surprisingly Met-Arg was partially processed. These results indicate a strict preference of the insulinoma prohormone endoprotease(s) for paired basic amino acids ending in arginine, but that processing efficiency of some sequences may be modulated by location within the precursor molecule.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.     

Mono- and dibasic proteolytic cleavage sites in insect neuroendocrine peptide precursors

Regulatory peptides are synthesized as part of larger precursors that are subsequently processed into the active substances. After cleavage of the signal peptide, further proteolytic processing occurs predominantly at basic amino acid residues. Rules have been proposed in order to predict which putative proteolytic processing sites are actually used, but these rules have been established for vertebrate peptide precursors and it is unclear whether they are also valid for insects. The aim of this paper is to establish the validity of these rules to predict proteolytic cleavage sites at basic amino acids in insect neuropeptide precursors. Rules describing the cleavage of mono- and dibasic potential processing sites in insect neuropeptide precursors are summarized below. Lys-Arg pairs not followed by an aliphatic or basic amino acid residue are virtually always cleaved in insect regulatory peptide precursors, but cleavages of Lys-Arg pairs followed by either an aliphatic or a basic amino acid residue are ambiguous, as is processing at Arg-Arg pairs. Processing at Arg-Lys pairs has so far not been demonstrated in insects and processing at Lys-Lys pairs appears very rare. Processing at single Arg residues occurs only when there is a basic amino acid residue in position -4, -6, or -8, usually an Arg, but Lys or His residues work also. Although the current number of such sites is too limited to draw definitive conclusions, it seems plausible that cleavage at these sites is inhibited by the presence of aliphatic residues in the +1 position. However, cleavage at single Arg residues is ambiguous. When several potential cleavage sites overlap the one most easily cleaved appears to be processed. It cannot be excluded that some of the rules formulated here will prove less than universal, as only a limited number of cleavage sites have so far been identified. It is likely that, as in vertebrates, ambiguous processing sites exist to allow differential cleavage of the same precursor by different convertases and it seems possible that the precursors of allatostatins and PBAN are differentially cleaved in different cell types. Arch. Insect Biochem. Physiol. 43:49-63, 2000.     

Effect of amino acid substitutions at the signal peptide cleavage site of the Escherichia coli major outer membrane lipoprotein

The requirement for the glycine residue at the COOH terminus of the signal peptide of the precursor of the major Escherichia coli outer membrane lipoprotein was examined. Using oligonucleotide-directed site-specific mutagenesis, this residue was replaced by residues of increasing side chain size. Substitution by serine had no effect on the modification or processing of the prolipoprotein. Substitution by valine or leucine resulted in the accumulation of the unmodified precursor, whereas threonine substitution resulted in slow lipid modification and no detectable processing of the lipid modified precursor. The results indicate that serine is the upper limit on size for the residue at the cleavage site. Larger residues at this position prevent the action of both the glyceride transferase and signal peptidase II enzymes, indicating that the cleavage site residue plays a role in events prior to proteolytic cleavage. The upper limit on size of the cleavage site residue is similar to that found for exported proteins cleaved by signal peptidase I, as well as eucaryotic exported proteins. The possibility that the cleavage site residue may have a role other than active site recognition by the signal peptidase is discussed.     

Processing and sorting of the prohormone convertase 2 propeptide

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.     

Sequence requirements for prohormone processing in mouse pituitary AtT-20 cells. Analysis using prorenins as model substrates

Although cleavage of peptides at sites marked by paired basic amino acids is a common feature of prohormone processing, little is known about the properties of endoprotease(s) responsible for cleavage of the precursor. To examine the cleavage specificity of a processing endoprotease, we have altered the Lys-Arg cleavage site of human prorenin to Arg-Arg, Lys-Lys and Arg-Lys by site-directed mutagenesis, and expressed the native and mutated precursors in mouse pituitary AtT-20 cells which are known to process foreign prohormones, including prorenin, at paired basic sites during the regulated secretory process. All native and mutated human prorenins were sorted into the regulated secretory pathway. The mutated precursor with Arg-Arg instead of the Lys-Arg native pair was processed at about half the efficiency of the native one, while the Lys-Lys and Arg-Lys mutants were not processed. Rat prorenin, which naturally has a Lys-Lys pair, was not processed in the cells. In addition, mouse Ren2 prorenin, which has a Ser residue next to the Lys-Arg pair, but not mouse Ren1 prorenin, which has a Pro residue next to the pair, was processed. These results suggest that the Arg residue at the COOH side of the basic pair is essential for cleavage of prorenins by a processing enzyme during the regulated secretory process in AtT-20 cells, although the NH2-side Lys residue also plays a role. The results also demonstrate that the processing enzyme cannot cleave the Arg-Pro peptide bond.     

Golgi/granule processing of peptide hormone and neuropeptide precursors: a minireview

Proteolytic processing of precursor proteins is a phylogenetically ancient and widely used mechanism for producing biologically active peptides. Proteolytic cleavage of proproteins begins only after transport to the Golgi apparatus has been completed and in most systems may continue for many hours within newly formed secretory vesicles as these are stored in the cytosol or transported along axons to more peripheral sites of release. Paired basic residues are required for efficient proteolysis in most precursors, suggesting that a small number of specialized tryptic proteases exist that have great site selectivity but can process many sites within the same precursor or in different precursors within the same cell, or in different cells or tissues. Cleavage-site choice may be strongly influenced by other factors, such as secondary and tertiary structure, but definitive structural information on precursor proteins is lacking. Modifications such as glycosylation, phosphorylation, and sulfation also are Golgi associated but are not known to influence proteolytic processing patterns. Golgi/granule processing also rarely occurs at sites other than pairs of basic amino acids, including single basic residues ( trypsinlike ), Leu-Ala, Leu-Ser, or Tyr-Ala bonds ( chymotrysinlike ) as well as other specialized nontryptic cleavages, suggesting that mixtures of proteases coexist in the Golgi/granule system. Cathepsin B-like thiol proteases, or their precursors, have been implicated as the major processing endopeptidases in several systems. Carboxypeptidase B-like enzymes also have been identified in secretion granules in several tissues and appear to be metalloenzymes similar in mechanism to the pancreatic carboxypeptidases, but with a lower pH optimum. The role of the Golgi apparatus in sorting newly formed secreted products from lysosomal hydrolases may have permitted the development in evolution of an intimate relationship between certain of the lysosomal degradative enzymes, such as cathepsin B or its precursors, and the Golgi/granule processing systems. The sequestration of the proteolytic products of precursors within secretion granules leads to the coordinate discharge of highly complex mixtures of peptides having related or overlapping biological activities. The cosecretion of nonfunctional peptide " leftovers ," such as the proinsulin C-peptide, can serve as useful markers of secretion or cellular localization, as well as of evolutionary relation ships. Errors in cleavage due to point mutations in precursors have been identified in several systems, leading to the accumulation of incorrectly processed materials in the circulation. These and/or defects (ABSTRACT TRUNCATED AT 400 WORDS)     

Improved efficiency of site-specific copper(II) ion-catalysed protein cleavage effected by mutagenesis of cleavage site

The peptide sequence (N)DKTH(C) was previously investigated as a site for efficient, specific cleavage of a fusion protein by cupric ions using a humanized gamma1 Fab' as a model protein. Here we show that conservative mutations to three of the residues in the introduced cleavage site resulted in cleavage sites that were significantly improved. They were cleaved more efficiently by Cu(2+), such that cleavage reactions could be shorter, of lower pH or at a lower temperature. Some were even found to be measurably cleaved by Ni(2+). Use of these new cleavage sequences along with cupric ions may provide a more rapid and less harsh method for cost-effective, large-scale proteolytic cleavage of fusion proteins and peptides.     

Expression of human pancreatic polypeptide in heterologous cell lines

Pancreatic polypeptide (PP) is initially synthesized as a larger precursor that requires post-translational processing to produce the biologically active hexatriacontapeptide. These steps include tryptic cleavage at paired basic residues, their subsequent removal by a carboxypeptidase B-like enzyme, and formation of a carboxyl-terminal amide moiety via the action of peptidyl-glycyl alpha-amidating monooxygenase. To examine these reactions further, we utilized the pZIPneo(SVX) retroviral vector to express a cDNA clone encoding human PP in several cell lines including a fibroblast line (psi-2), two endocrine cell lines known to produce amidated peptides (AtT-20 and PC12), and two lines that do not ordinarily produce amidated peptides (RIN5-f and GH3). Transfected psi-2 cells produced an unprocessed precursor of PP that appeared to be secreted constitutively with little remaining in intracellular stores. No post-translational processing of the PP precursor was evident in these cells. By contrast, all 4 endocrine-derived cell lines, regardless of the nature of their endogenous products, were capable of expressing fully processed and carboxyl-terminally amidated PP. Moreover, these lines had the ability to store the processed products. Our results support the notion that post-translational processing of peptide hormone precursors requires storage in secretory granules that contain the appropriate processing enzymes. Furthermore, enzymes such as peptidyl-glycyl alpha-amidating monooxygenase that are required for processing peptides may be a common feature of endocrine-derived cells regardless of the requirement for their activity to process endogenous products.     

Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.     

A putative processing enzyme for proenkephalin in bovine adrenal chromaffin granule membranes. Purification and properties

A putative processing enzyme for proenkephalin, with activity directed toward basic residues, was purified over 2000-fold from washed bovine adrenal medullary chromaffin granule membranes. The molecular mass of this membrane-bound adrenal trypsin-like enzyme (mATLE) is 31 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the enzyme is extremely basic, binding to carboxymethyl-Sephadex at pH 8.5. The pH optimum of mATLE using t-butoxycarbonyl-Glu-Lys-Lys-aminomethylcoumarin as a substrate is 8.5-8.7, and its Km value for this substrate is 2.2 mM. mATLE activity was inhibited by soybean trypsin inhibitor, lima bean trypsin inhibitor, and aprotinin but not by metal chelators or thiol-directed reagents. Sequencing of cleavage products released from Peptide B revealed that the enzyme preferentially cleaves between and following the paired basic residues at positions 23 and 24 of Peptide B (thus generating [Met-enkephalin]-Arg-Phe and Arg-[Met-enkephalin]-Arg-Phe). Dynorphin A was cleaved following a single lysine at position 11 but not at the paired arginine site. Our results suggest that mATLE is a trypsin-like serine protease with the specificity appropriate to that of a proenkephalin processing enzyme.