Paired Basic Residues Research Articles

Evidence for distinct dibasic processing endopeptidases with Lys-Arg and Arg-Arg specificities in neurohypophysial secretory granules

Two Ca 2+-dependent endopeptidases endowed with specificities for paired basic residues have been disclosed in rat and ox neurohypophysial secretory granules. Specificities investigated by using synthetic fluorogenic substrates showed the presence of a Lys-Arg endopeptidase with optimum pH close to the granule pH (5.5) and of an Arg-Arg endopeptidase more active at pH 7.0. Granule extracts have virtually no activity towards Lys-Lys-containing susbtrate or monobasic substrates. Pro-Gly-Lys-Arg-chloromethylketone appears a very efficient inhibitor for the Lys-Arg enzyme. Soluble and membrane-bound forms of both endopeptidases have been detected. pH-dependence of membrane binding and partitioning into Triton X-114 suggest that the membrane-bound form of Lys-Arg endopeptidase is associated through an amphiphilic α-helix. It is proposed that the enzyme Lys-Arg cleaves prooxytocin and provasopressin at their signal sequence Gly-Lys-Arg when these precursors arrive in the neurosecretory granules. The processing proceeds in the granules through carboxypeptidase E and α-amidating enzyme complex for giving mature pharmacologically active nonapeptide hormones.

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

Minireview: The New Eukaryotic Precursor Processing Proteinases

One of the most exciting developments in the field of neuroendocrinology was the discovery last year that mammalian cells contain specialized proteinases homologous to the bacterial subtilisins. In contrast to the bacterial subtilisins, which show little sequence specificity, these new mammalian proteinases are remarkably specific and cleave exclusively at certain sets of basic residues. It now appears likely that this family of proteinases is responsible for the proteolytic activation of prohormones, neuropeptide precursors, and in all likelihood a wide variety of other protein precursors. The fact that yeasts use a proteinase known as Kex2 to process the precursor to the α-factor pheromone has been accepted since the mid-80s (see review, Ref. 1). Since this yeast precursor is cleaved at pairs of basic residues, similar to the sites at which proinsulin as well as many other mammalian hormone and neuropeptide precursors are cleaved, it was long hypothesized that mammalian cells contain a homologous proteinase which is responsible for the paired basic cleavage event. Many laboratories tried without success to clone such mammalian enzymes from cDNA libraries using Kex2-based probes.

Coordinate regulation of mRNA levels of pro-opiomelanocortin and the candidate processing enzymes PC2 and PC3, but not furin, in rat pituitary intermediate lobe

Pro-opiomelanocortin is a multivalent hormone precursor which is processed at pairs of basic residues in a tissue-specific manner to release biologically active peptides. We have examined the message levels of three candidate pro-opiomelanocortin processing enzymes in the intermediate lobe of the rat pituitary following treatment with a dopamine receptor agonist and antagonist which are known to regulate pro-opiomelanocortin mRNA levels. Message levels for PC2 and PC3 but not furin were coordinately regulated with pro-opiomelanocortin transcripts supporting a role for PC2 and PC3 in the maturation of the pro-opiomelanocortin precursor in the rat pituitary intermediate lobe.

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.

Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake.

We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media. This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions. Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction. Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2). The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells. One such factor could be the mammalian equivalent of the gene product of UPC2.

Isolation and functional expression of a mammalian prohormone processing enzyme, murine prohormone convertase 1.

We have combined gene cloning with an assay for prohormone biosynthesis and processing in Xenopus oocytes to identify the genes that encode mammalian prohormone processing enzymes. The coinjection of RNA encoding murine prohormone convertase 1 (mPC1), a mammalian endoprotease, along with proopiomelanocortin RNA into an oocyte results in the appropriate cleavage after paired basic residues in the proopiomelanocortin polyprotein necessary to generate corticotropin. The ability of mPC1 to generate corticotropin, along with the observation that mPC1 is specifically expressed in endocrine and neuronal cells, suggests that the mPC1 gene encodes the endopeptidase responsible for the pathway of proopiomelanocortin cleavage observed in the anterior pituitary.

Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.

Expression and Sorting of Rat Plasma Kallikrein in POMC-Producing AtT-20 Cells

A vaccinia virus (VV) vector was used to express rat plasma kallikrein (rPK) in the constitutively secreting cells, BSC-40, and in the endocrine regulated cells, AtT-20. Using a specific rPK antibody and a fluorogenic substrate, Phe-Phe-Arg-AMC, we demonstrated that in both cell lines VV infections resulted in the synthesis of an immunoreactive enzyme predominantly present as a zymogen which can be activated with trypsin. Stimulation of VV:rPK-infected AtT-20 cells with either 5mM 8-bromo-cAMP or 56 mM KCl resulted in a different pattern of rPK and ACTH secretion, strongly suggesting that rPK follows the constitutive secretory pathway. Finally, the 10% rPK activity found within AtT-20 cell extracts had no effect on pro-opiomelanocortin (POMC) processing either intracellularly or extracellularly. The above data show that the biosynthetic machinery of both cell lines analyzed does not allow the efficient activation of plasma prekallikrein. Finally, despite the PK's demonstrated ability to cleave various hormone precursors in vitro at pairs of basic residues, in vivo, we did not obtain evidence that this hepatic enzyme can also act as an intracellular pro-protein processing enzyme.

Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae.

The Kex2 protein of the yeast Saccharomyces cerevisiae is a membrane-bound, Ca2(+)-dependent serine protease that cleaves the precursors of the mating pheromone alpha-factor and the M1 killer toxin at pairs of basic residues during their transport through the secretory pathway. To begin to characterize the intracellular locus of Kex2-dependent proteolytic processing, we have examined the subcellular distribution of Kex2 protein in yeast by indirect immunofluorescence. Kex2 protein is located at multiple, discrete sites within wild-type yeast cells (average, 3.0 +/- 1.7/mother cell). Qualitatively similar fluorescence patterns are observed at elevated levels of expression, but no signal is found in cells lacking the KEX2 gene. Structures containing Kex2 protein are not concentrated at a perinuclear location, but are distributed throughout the cytoplasm at all phases of the cell cycle. Kex2-containing structures appear in the bud at an early, premitotic stage. Analysis of conditional secretory (sec) mutants demonstrates that Kex2 protein ordinarily progresses from the ER to the Golgi but is not incorporated into secretory vesicles, consistent with the proposed localization of Kex2 protein to the yeast Golgi complex.

Initial characterization of multiple endopeptidases in bovine adrenal chromaffin vesicles

All regulatory peptides are synthesized as large, inactive precursor proproteins that must undergo specific endoproteolytic processing to yield bioactive peptides. In most cases, enzymatic release of the biologically active peptides occurs by endoproteolytic cleavage at doublets of basic amino acid residues that precede and/or follow that particular sequence [1,2]. The catecholamine-containing chromaffin vesicles of the adrenal medulla are enriched in a great variety of regulatory peptides (i.e. enkephalins, neurotensin, neuropeptide Y, etc.) and thus are good sources for the isolation and characterization of peptide processing enzymes [3,4,5]. To isolate putative endopeptidases of the thioland serineprotease families, the dialysed lysate of purified bovine chromaffin vesicles was consecutively fractionated through p-chloro-mercuribenzoateagarose (PCMB-agarose), p-aminobenzamidineagarose (p-ABZ-agarose) and soybean trypsin inhibitor-agarose (STI-agarose) affinity columns. Three intermediate proenkephalin precursor peptides (BAM12P, BAM22P and amidorphin) were used as substrates for the assay of endopeptidase activities. These peptides contain pairs of basic amino acids, Arg-Arg, Lys-Arg and Lys-Lys, which have putative cleavage sites for the endopeptidases. Degradation peptide fragments were separated by reverse phase HPLC and identified by FAB mass spectrometry, amino acid analysis and sequence analysis, which was performed with an Applied Biosynthesis model 477a protein sequencer using Edman degradation chemistry. The fraction retained and eluted from PCMB-agarose affinity chromatography hydrolyzed the Arg-Arg sequence of BAM12P, resulting in the generation of Met-enkephalin and Met-enkephalin-Arg at pH 5.7. However, this enzyme preparation was unable to hydrolyze amidorphin at the Lys-Lys pair of basic residues. This activity was inhibited by PCMB and E64, indicating that a thiol protease is involved. The dialysate fraction that was not retained by the PCMB-agarose column was subsequently retained and eluted from the p-ABZagarose affinity column. This dialysate fraction contained enzyme activity which cleaved at the Lys-Arg of BAM22P and at the Lys-Lys of amidorphin at pH 7.4. BAM12P was however a poor substrate for this fraction. This activity was not inhibited by ST1, which is indicative of a non-trypsin-like endopeptidase. Additionally, a separate endopeptidase cleaving at Glu-Trp of BAM22P, resulting in the generation of BAM12P, was also found in this preparation. The dialysate fraction not retained in the first two columns but retained and eluted from the STI-agarose affinity column had an enzyme activity capable of hydrolyzing amidorphin at the carboxy side of Lys-Lys. This activity was completely inhibited by STI which is indicative of a trypsin-like endopeptidase. BAM12P however was poorly cleaved by this preparation. This study demonstrates that a variety of different endopeptidase activities is found in soluble lysates of adrenal medulla chromaffin vesicles. A multiplicity of peptide processing enzymes with different specificities suggests the possibility that modification of a particular processing enzyme may result in specific changes in the cocktail of regulatory peptides.

Proteolysis in Rat Hypothalamic Neurosecretory Granules: Characterization of an a-Chymotrypsin-Like Activity in the Pathway of Intracellular Processing of Prohormones*

To yield biologically active hormones, prohomones are processed by cleavages at paired basic amino acid residues. In this study we report that a chymotrypsin-like activity has been colocalized with trypsin-like and carboxypeptidase-B-like proteases in neurosecretory granules, the site of intracellular processing of prohomones. Using a peptide of 11 amino acids as a simple model system for the study of prohomone processing, we have identified in neurosecretory granule membranes a novel protease that specifically recognizes and cleaves peptide bonds at aromatic residues. Studies were also performed with the fluorogenic peptide substrate N-succinyl-leucyl-leucyl-valyl-tyrosine-7-amino-4-methyl-coumarine . The identified protease activity is inactivated by a cloromethyl ketone derivative (Tos-Phe-CH2-Cl) by Trasylol, and markedly by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. This activity is colocalized with other prohomone-processing enzymes in neurosecretory granules, which indicates that this activity is involved in the prohomone processing. Alternatively, this activity may function in the degradation of homones and neuropeptides when they are released in synapses.

Primary structure of the precursor for the sea anemone neuropeptide Antho-RFamide (less than Glu-Gly-Arg-Phe-NH2).

Neuropeptides containing the carboxylterminal sequence Arg-Phe-NH2 are found throughout the animal kingdom and are important substances mediating neuronal communication. Here, we have cloned the cDNA coding for the precursor protein of the sea anemone neuropeptide (Antho-RFamide) less than Glu-Gly-Arg-Phe-NH2. This precursor is 334 amino acids in length and contains 19 copies of unprocessed Antho-RFamide (Gln-Gly-Arg-Phe-Gly), which are tandemly arranged in the C-terminal part of the protein. Paired basic residues (Lys-Arg) or single basic residues (Arg) occur at the C-terminal side of each Antho-RFamide sequence. These are likely signals for posttranslational cleavage. The processing signals at the N-terminal side of each Antho-RFamide sequence, however, include acidic residues. Processing at these amino acids must involve either an amino- or an endopeptidase that cleaves C-terminally of aspartic acid or glutamic acid residues. Such processing is, to our knowledge, hitherto unknown for peptidergic neurons. The Antho-RFamide precursor also contains two copies of the putative Antho-RFamide-related peptide Phe-Gln-Gly-Arg-Phe-NH2 and one copy of Tyr-Val-Pro-Gly-Arg-Tyr-NH2. In addition, the precursor protein harbors four other putative neuropeptides that are much less related to Antho-RFamide. This report shows that the biosynthetic machinery for neuropeptides in coelenterates, the lowest animal group having a nervous system, is already very efficient and similar to that of higher invertebrates, such as mollusks and insects, and vertebrates.

Specific cleavages of arginyl peptide bonds at basic amino acid pairs by a serine proteinase from the microsomal membranes of rat liver

The specificity of action of a serine proteinase from the microsomal membranes of rat liver was investigated at pH 7.5 and 37°C using various peptides as substrates. HPLC analyses of the peptides produced followed by their amino acid analyses have revealed that the enzyme is a unique endopeptidase specifically cleaving arginyl peptide bonds at paired basic amino acid residues. Thus, the enzyme is suggested to be a kind of processing proteinase involved in the conversion of proproteins to their mature forms. Indeed, the enzyme cleaved specifically the NH 2-terminal 20-residue peptide of proalbumin at the Arg-Arg sequence.

Prohormone Processing by Yeast Proteases

Investigations of the precursors of alpha-pheromone and killer toxin in the yeast Saccharomyces cerevisiae have defined the genes coding (KEX1 and KEX2) for the proteases which are responsible for their processing. In addition to processing at pairs of basic residues it is evident that yeast can also process at monobasic sites. We present data on the Kex1p and Kex2p enzymes, their cellular localization, and their post-translational modification. In addition initial characterisation of the monobasic specific protease and the isolation of mutants defective in this activity are presented. The use of the yeast system as a model for the processing of mammalian prohormones is discussed.

Furin: The Prototype Mammalian Subtilisin-Like Proprotein-Processing Enzyme

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.

Evolution of the insulin superfamily: cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus.

Although insulin and the insulin-like growth factors (IGFs) share marked similarities in amino acid sequence and biological activity, their evolutionary origins have not been resolved. To investigate this issue, we recently cloned a cDNA encoding an insulin-like peptide (ILP) from a primitive chordate species, amphioxus (Branchiostoma californiensis). The deduced sequence of amphioxus preproILP indicates that it is a hybrid molecule containing features characteristic of both insulin and IGF. Like proinsulin, amphioxus proILP contains a C-peptide, which is flanked by paired basic residues and is probably removed by proteolysis. However, proILP also contains an extended carboxyl-terminal peptide region that can be divided into D and E domains similar to those of proIGF. Sequence comparisons show that the amphioxus ILP A and B domains are equally homologous to those of human insulin and IGF-I and -II. Based on these results and the exon-intron organization of the amphioxus ILP gene, we propose that IGF emerged at a very early stage in vertebrate evolution from an ancestral insulin-type gene.

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

Expression of spasmolysin (FIM-A.1): An integumentary mucin from Xenopus laevis

In the past, a unique type of precursor for a secretory protein was discovered. It contains a central repetitive domain rich in threonine residues and terminal cysteine-rich domains. Due to striking homologies of these terminal domains with pancreatic spasmolytic polypeptide, originally the name “prepro-spasmolysin” was proposed. Here we show that the mature protein has a MW of about 130 kDa, consisting of about 70% carbohydrate and 30% protein. Similar O-linked glycoproteins have been found in mucins from human intestine. For this and numerous other reasons we decided to rename this glycoprotein “frog integumentary mucin A.1” (FIM-A.1). Furthermore, analysis of the protein with specific antibodies against the predicted C-terminal end indicates that FIM-A.1 is probably not processed at pairs of basic residues. In situ hybridization as well as immunofluorescence studies revealed that FIM-A.1 is expressed and stored exclusively in mature mucous glands of Xenopus laevis skin. Only cone cells at the proximal part of these glands do not synthesize FIM-A.1. In contrast, all other physiologically active peptides from X. laevis skin investigated so far are synthesized in granular glands. A hypothetical function of FIMs for defense against microbial infections is discussed.