A novel prohormone processing site in Aplysia californica: the Leu-Leu rule

Neuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non-basic sites. Cleavage at Leu-Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non-native synthetic peptides containing Leu-Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu-Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu-Leu residues. Products resulting from cleavage at Leu-Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity.     

Aplysia capsulin is localized to egg capsules and egg cordon sheaths and shares sequence homology with Drosophila dec-1 gene products

Differential library screening of an albumen gland cDNA library, Western blot analysis, protein expression, immunolocalization studies, comparative genomics, and secretion assays identified a major Aplysia californica albumen gland protein ('capsulin') that is localized to egg capsules and to the sheaths of the egg cordon. Capsulin shared sequence homology with eggshell proteins encoded by the Drosophila dec-1 gene. The 1790-amino acid A. californica precursor contains 17 repeat sequences that are flanked by basic residue processing sites. The numerous proteolytic processing sites may facilitate the breakdown of capsulin prior to when veliger larvae break out of egg capsules as free-swimming larvae. An Aplysia brasiliana capsulin repeat sequence was 97% identical to its A. californica homolog. Capsulin fragments were not detected in the eluates of egg cordons, suggesting that capsulin is not a candidate water-borne pheromone precursor.     

Processing of the egg-laying hormone (ELH) precursor in the bag cell neurons of Aplysia

Egg laying in Aplysia is mediated by a battery of neuropeptides released from the bag cell neurons. Predominant intermediates in the proteolytic processing of the Aplysia egg-laying hormone neuropeptide precursor were characterized using biochemical and immunological techniques. Following removal of the signal peptide, a rapid cleavage at the tetrabasic sequence Arg-Arg-Lys-Arg separates the amino and carboxyl regions of the prohormone. Processing of the carboxyl-terminal portion of the precursor then proceeds rapidly via two further cleavages at dibasic residues, resulting in a well defined product mixture within 4 h of chase. By contrast, processing of the amino-terminal side of the molecule proceeds only partially to completion after 20 h of chase and a well defined set of intermediates is not observed. Molecular genetic, physiological, and behavioral studies in conjunction with the biochemical investigations presented here are defining the information flow which governs the egg-laying behavior of Aplysia.     

The processing of peptide precursors. 'Proline-directed arginyl cleavage' and other monobasic processing mechanisms

The classical conversion site in precursors of regulatory peptides is a sequence of two basic amino acids. During recent years, however, a group of monobasic cleavage sites has emerged. In certain cell systems it has been shown that the monobasic cleavage mechanism is both a specific mechanism which only attacks a particular basic residue, and a distinct mechanism which can be separated from the dibasic cleaving mechanism within the same cell. The vast majority of monobasic cleavages occur at single arginines although cleavage after a lysine residue has also been demonstrated. There is no 'consensus sequence' of amino acids surrounding the single basic residue which is the apparent signal for proteolytic processing. However, in approximately one third of the cases, a proline residue is found either just before or just after the basic residue. On the basis of this 'proline-directed arginyl cleavage' it is discussed how the conformation of the peptide backbone might be important for this type of cleavage. Finally, it is suggested that tissue-specific expression of different processing enzymes, e.g. dibasic and monobasic specific forms, might explain the tissue-specific processing of precursors like the pro-opiomelanocortin and the CKK and somatostatin precursor.     

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.     

A putative processing enzyme from Aplysia that cleaves dynorphin A at the single arginine residue

A peptidase activity cleaving at single arginine residues has been detected in extracts of the atrial gland of Aplysia Californica. The enzyme assay consisted of incubation of enzyme with the mammalian opioid peptide dynorphin A and detection by specific radioimmunoassay of dynorphin (1-8), a single arginine cleavage product. The peptidase activity was characterized following chromatography on DEAE-cellulose. Activity was abolished by a thiol-directed inhibitor and chelators and activated by dithiothreitol and cobalt chloride. The pH optimum was 6.2 in phosphate buffer. Analysis of the products of two substrates suggested that cleavage was occurring on the amino side of the arginine residue.     

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.     

Serotonin induces selective cleavage of the PKA RI subunit but not RII subunit in Aplysia neurons

PKA type I and type II are activated in Aplysia neurons by stimulation with serotonin (5-HT), which causes long-term facilitation (LTF). The proteolysis of the regulatory subunit (R) is thought important for the persistent activation of PKA, which is necessary to produce LTF. In this study, we report that the type I regulatory subunit (RI) and type II regulatory subunit (RII) are differentially regulated by proteolytic cleavage. RI, but not RII, was selectively cleaved after 5-HT treatment for 2h in Aplysia neurons. Interestingly, the proteasome inhibitor MG132 inhibited the cleavage of RI caused by 5-HT treatment in Aplysia neuron. Besides extracts from Aplysia ganglia treated with 5-HT cleaved (35)S-labeled RI synthesized in vitro, but not (35)S-labeled RII. This suggests that 5-HT induces the activation state of RI-specific proteolytic cleavage.     

Expression and processing of mouse proopiomelanocortin in bovine adrenal chromaffin cells. A model system to study tissue-specific prohormone processing.

Many neuroendocrine precursor proteins, such as proopiomelanocortin (POMC), are cleaved in a tissue specific manner at distinct pairs of basic amino acids. Elucidating the specificity of the prohormone endoprotease(s) is essential to understanding cleavage specificity. However, isolation of these enzymes has been difficult, due to the inability to distinguish authentic maturation enzyme from the many other trypsin-like activities present in tissue homogenates. Recently, a "signature" of the insulin cell endoprotease(s) was defined in vivo by assessing the processing of a series of mutant cleavage sites in a model prohormone, mouse POMC (mPOMC) (Thorne, B. A., and Thomas, G. (1990) J. Biol. Chem. 265, 8436-8443. To investigate mechanisms of tissue-specific processing, we sought to identify the endoprotease signature of a cell having a processing phenotype distinct from insulinoma cells. In this report, the cleavage site specificity of the endoprotease(s) expressed in bovine adrenal chromaffin cells is examined. High levels of mPOMC (1.6 pmol/10(6) cells) were expressed in these cells using a vaccinia virus vector, and the precursor was targeted to the regulated secretory pathway. Analysis of POMC-derived peptides revealed that chromaffin cells processed the prohormone to a set of peptides highly similar to anterior pituitary corticotrophs, including adrenocorticotropin hormone (ACTH) and beta-lipotropin, gamma-lipotropin, and beta-endorphin. This processing contrasted with the pattern of cleavage site utilization in Rin m5F insulinoma cells, which more closely resembled that of the intermediate pituitary melanotrophs. However, the processing preference for the sequences of pairs of basic amino acids (as tested using the entire series of mutant cleavage sites; -LysArg- (native), -ArgArg-, -ArgLys-, -LysLys-, -HisArg-, -MetArg- at the ACTH/beta-lipotropin junction and -LysLys- (native), -LysArg-, -ArgArg-, -ArgLys- in beta-endorphin) was the same in both insulinoma and adrenal chromaffin cells, suggesting recognition and cleavage by similar enzymes in both cell types. The cell-specific processing of mPOMC may thus result from expression of a common core set of processing enzymes and factors unique to each cell type affecting the enzyme accessibility to precursor cleavage sites.     

Utilization of Escherichia coli outer-membrane endoprotease OmpT variants as processing enzymes for production of peptides from designer fusion proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp(97) is proposed to interact with the P1' amino acid of its substrates, OmpT variants with variations at Asp(97) were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1'). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg downward arrow motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Endopeptidases and prohormone processing

Peptide hormones and peptide transmitters are generated from polypeptide precursors by specific cleavage reactions which take place principally at sites formed by single or paired basic residues. Not all the possible cleavage sites are utilised, however, and the degree of processing of many propeptides has been found to vary according to the tissue of origin. The restricted nature of processing reactions could point to the existence of a series of enzymes with stringent specificities, recognising regions of structure in addition to the single or paired basic residues. Alternatively the action of processing enzymes may be directed by conformation of the pro-peptide which could focus the action of a protease onto or away from a particular site. In addition certain post-translational modifications such as glycosylation or phosphorylation may influence the accessibility of a site to the approach of a processing enzyme. In this review we describe recent advances that have been made in the characteristisation of proteolytic processing enzymes, we examine the relevance of the various factors that could account for restricted processing and discuss new approaches that may lead to better understanding of the mechanisms involved.     

Proteolytic processing of a peptide precursor in Aplysia neuron R14

The large neurons of the mollusc Aplysia are useful for studying the biogenesis of neuropeptides in single cells. Neuron R14 in the abdominal ganglion synthesizes large quantities of a 10-kDa neuropeptide precursor. The amino acid sequence of this precursor has been defined by analysis of the nucleotide sequence of a cDNA clone. We labeled proteins in vivo by microinjection of radioactive amino acids into individual R14 neurons. The labeled peptides were fractionated by high performance liquid chromatography and subjected to Edman degradation, thus enabling us to determine post-translational processing sites. Cleavage of the signal sequence was observed and at two internal sites. Cleavage at these internal sites occurs at basic amino acids and results in three products, a 2.9-, a 4.9-, and a 1.4-kDa peptide. These studies of protein processing serve as a basis for further investigations of the biogenesis and physiological activities of the neuropeptides.     

p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.     

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.     

Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp⁹⁷ is proposed to interact with the P1′ amino acid of its substrates, OmpT variants with variations at Asp⁹⁷ were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1′). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg↓motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 A resolution

The binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X-ray crystallography. The three-dimensional structure of the complex has been refined at 1.9 A resolution to a crystallographic R-factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co-ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side-chain of Arg66 (E10) which, from an outer orientation in the 'aquo-met' ligand-free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.