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.     

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.     

Proteolysis of ProPTHrP(1-141) by "prohormone thiol protease" at multibasic residues generates PTHrP-related peptides: implications for PTHrP peptide production in lung cancer cells

The parathyroid hormone-related protein (PTHrP) precursor requires proteolytic processing to generate PTHrP-related peptide products that possess regulatory functions in the control of PTH-like (parathyroid-like) actions and cell growth, calcium transport, and osteoclast activity. Biologically active peptide domains within the PTHrP precursor are typically flanked at their NH2- and COOH-termini by basic residue cleavage sites consisting of multibasic, dibasic, and monobasic residues. These basic residues are predicted to serve as proteolytic cleavage sites for converting the PTHrP precursor into active peptide products. The coexpression of the prohormone processing enzyme PTP ("prohormone thiol protease") in PTHrP-containing lung cancer cells, and the lack of PTP in cell lines that contain little PTHrP, implicate PTP as a candidate processing enzyme for proPTHrP. Therefore, in this study, PTP cleavage of recombinant proPTHrP(1-141) precursor was evaluated by MALDI mass spectrometry to identify peptide products and cleavage sites. PTP cleaved the PTHrP precursor at the predicted basic residue cleavage sites to generate biologically active PTHrP-related peptides that correspond to the NH2-terminal domain (residues 1-37) that possesses PTH-like and growth regulatory activities, the mid-region domain (residues 38-93) that regulates calcium transport, and the COOH-terminal domain (residues 102-141) that modulates osteoclast activity. Lack of cleavage at other types of amino acids demonstrated the specificity of PTP processing at basic residue cleavage sites. Overall, these results demonstrate the ability of PTP to cleave the PTHrP precursor at multibasic, dibasic, and monobasic residue cleavage sites to generate active PTHrP-related peptides. The presence of PTP immunoreactivity in PTHrP-containing lung cancer cells suggests PTP as a candidate processing enzyme for the PTHrP precursor.     

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.     

Cellular peptide processing after a single arginyl residue. Studies on the common precursor for pancreatic polypeptide and pancreatic icosapeptide

The processing of the common precursor for pancreatic polypeptide and pancreatic icosapeptide was studied in primary cultures of endocrine cells isolated from the duodenal part of the canine pancreas. Biosynthetically labeled peptides were characterized by enzymatic digestion and radiosequencing and compared to a COOH-terminally extended form of the icosapeptide which was isolated from canine pancreas and also sequenced. It was substantiated that, in these cell cultures, processing can be studied at a classical dibasic site between the pancreatic polypeptide and the icosapeptide, and at a monobasic processing site between the icosapeptide and its COOH-terminal extension. Pulse-chase experiments showed that the monobasic cleavage occurs later than the dibasic one in the biosynthetic process; the monobasic site was apparently not cleaved before the prohormone had been processed at the dibasic site. The monobasic processing could also be distinguished from the dibasic cleavage mechanism as, in time, the cells gradually lost the ability to cleave at the monobasic site while the dibasic processing was unaffected. It is concluded that monobasic conversion, which is important in the activation of a series of hormones, neuropeptides, and growth factors, is a distinct cellular processing mechanism.     

Endoproteolytic processing of the human protein C precursor by the yeast Kex2 endopeptidase coexpressed in mammalian cells

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation, and beta-hydroxylation of specific amino acids and endoproteolytic processing to remove the pre- and propeptides as well as the pair of basic amino acids which connect the light and heavy chains in the precursor. Previous studies with a recombinant mammalian expression system have indicated that the endopeptidase in several mammalian cell types which recognizes and cleaves this dibasic site has a substrate specificity for sites which also include a basic amino acid in the -4 position (Foster et al., 1990). Since the human protein C precursor has His154 in the -4 position, it is poorly and incompletely cleaved in BHK and several other mammalian cell lines and also apparently secreted from the liver as a mixed population of mature two-chain and precursor one-chain molecules. In the present study, a mammalian expression system has been used to study the effect of coexpressing the protein C precursor together with the yeast Kex2 endopeptidase which is known to recognize and process dibasic pairs within peptide precursors in yeast. Coexpression of the KEX2 gene resulted in complete conversion of the protein C precursor to the mature two-chain form. Amino-terminal sequencing of the cleavage products has indicated that the cleavage occurs in the correct location and that this site is preferentially recognized by the yeast endopeptidase within the context of the mammalian cell secretory pathway.     

Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: effects of sequence alterations on efficiency of cleavage

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation and beta-hydroxylation of specific amino acids, and endoproteolytic processing to remove the pre- and propeptides and also to remove the pair of basic amino acids that connect the light and heavy chains in the precursor. Specific molecular signals have been elucidated which direct several of these modifications; however, the mechanism for cleavage and removal of the basic amino acid pair has not been established. In the present study, a recombinant mammalian expression system has been used to study the molecular signals that direct removal of this basic amino acid pair. Mutations were introduced by site-directed mutagenesis either to insert additional basic amino acids or to alter the sequence adjacent to the basic pair by point mutations. The mutant protein precursors were expressed and analyzed for the degree of processing to 2-chain form and also for the location of the cleavage site (by N-terminal sequencing) and subsequent removal of the basic amino acids from the newly formed C terminus of the light chain. These experiments have shown that human protein C can be readily synthesized and secreted in several mammalian cell lines. However, cell lines vary considerably in their capacity to remove the dibasic pair in the protein C precursor and, like the liver, secrete a mixed population of 1-chain and 2-chain forms of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

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.     

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.     

Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids.

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.     

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.     

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)     

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues

During the course of reversed-phase high-pressure liquid chromatography (RP-HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420-493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597-653 and located at the COOH-terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg-Lys-Lys, present at positions 594-596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.     

Effect of amino acid analogs on the processing of the pancreatic polypeptide precursor in primary cell cultures

Amino acid analogs, which can be incorporated into nascent peptide chains were used in cultures of endocrine cells from canine pancreas to study the effect on processing of the metabolically labeled precursor for pancreatic polypeptide. Analogs for basic amino acids, canavanine, and aminoethylcysteine prevented the di-basic processing of the prohormone. The polar leucine analog, beta-hydroxyleucine, only partially perturbed the function and cleavage of the signal peptide but efficiently and unexpectedly blocked the dibasic cleavage of the prohormone. Other nonbasic amino acid analogs, beta-hydroxynorvaline and azetidine-2-carboxylic acid, which only could be incorporated into the prohormone at a distance from the processing site, also prevented dibasic cleavage of the prohormone. Although there are no phenylalanine residues in the prohormone, analogs for this amino acid, fluoro-phenylalanine and particularly phenylserine, could also block the processing of the prohormone at the dibasic site. This effect was prevented by addition of a small quantity of phenylalanine. It is concluded that amino acid analogs can interfere with precursor processing through altering both the primary and the secondary structure of the precursor but also through incorporation into cosynthesized protein(s) which are necessary for the precursor processing.     

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.     

Evidence that differentiates between precursor cleavages at dibasic and Arg-X-Lys/Arg-Arg sites.

It is well known that precursor cleavage at paired basic amino acids (e.g., Lys-Arg, Arg-Arg) within the regulated secretory pathway is one of the key steps to produce bioactive peptides. On the other hand, we have recently shown that precursors with an Arg residue at the fourth residue upstream of the cleavage site besides the basic pair, i.e. with the Arg-X-Lys/Arg-Arg (RXK/RR) motif, are cleaved within the constitutive secretory pathway. To discriminate between the precursor cleavage at RXK/RR sites within the constitutive pathway and that at dibasic sites within the regulated pathway, we examined the effects of drugs affecting the secretory process, intracellular Ca2+ depletion, and a protease inhibitor on these cleavages. Chloroquine (a weak base), depletion of intracellular Ca2+ by A23187 (a Ca2+ ionophore), and the Pittsburgh-type mutant of alpha 1-protease inhibitor differentially affected these two cleavages. Brefeldin A, which impedes protein transport from the endoplasmic reticulum to the Golgi complex, inhibited both cleavages. Colchicine (an anti-microtubular drug) had no discernible effect on either cleavage. These observations support the notion that the precursor cleavages at dibasic and RXK/RR sites occur in different subcellular compartments, and are catalyzed by different processing endoproteases.     

Determinants for cleavage of the chlorophyll a/b binding protein precursor: a requirement for a basic residue that is not universal for chloroplast imported proteins

We demonstrate that the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP of Photosystem II), encoded by a Type I gene, contains distinct determinants for processing at two sites during in vitro import into the chloroplast. Using precursors from both pea and wheat, it is shown that primary site processing, and release of a approximately 26-kD peptide, depends on an amino-proximal basic residue. Substitution of an arginine at position -4 resulted in an 80% reduction in processing, with the concomitant accumulation of a high molecular weight intermediate. Cleavage occurred normally when arginine was changed to lysine. The hypothesis that a basic residue is a general requirement for transit peptide removal was tested. We find that the precursors for the small subunit of Rubisco and Rubisco activase do not require a basic residue within seven amino acids of the cleavage site for maturation. In the wheat LHCP precursor, determinants for efficient cleavage at a secondary site were identified carboxy to the primary site, beyond what is traditionally called the transit peptide, within the sequence ala-lys-ala-lys (residues 38-41). Introduction of this sequence into the pea precursor, which has the residues thr-thr-lys-lys in the corresponding position, converted it to a substrate with an efficiently recognized secondary site. Our results indicate that two different forms of LHCP can be produced with distinct NH2-termini by selective cleavage of a single precursor polypeptide.     

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159–66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl–lysyl–bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.