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PubMed2003

Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin.

Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.

Amino Acid Sequence↗
PubMed1999

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.

Aspartic Acid Endopeptidases↗
PubMed1999

Immunohistochemical distribution of the prohormone convertase PC5-A in rat brain.

Prohormone convertase 5 is an endoprotease of the kexin/subtilisin-like family, which has been postulated to play a role in the proteolytic maturation of a variety of pro-peptides in the mammalian brain. In order to gain insight into the functional role of prohormone convertase 5 in the central nervous system, the regional, cellular and subcellular distributions of the enzyme were investigated by immunohistochemistry in rat brain using an N-terminal-directed specific antibody shown previously to recognize both the mature and unprocessed forms of the enzyme. Throughout the brain, prohormone convertase 5 immunoreactivity was concentrated within nerve cell bodies and proximal dendrites. No prohormone convertase 5 immunoreactivity was associated with astrocytes, as confirmed by the absence of prohormone convertase 5 immunolabeling in cells immunopositive for the glial protein S-100alpha. Within neurons, prohormone convertase 5 immunoreactivity was concentrated within the Golgi apparatus, as revealed immunohistochemically within the same sections using antibodies against the medial cisternae protein MG-160. It was also present within small vesicular-like elements distributed throughout the cytoplasm of perikarya and dendrites, but not of axons, as confirmed by its lack of co-localization with the synaptic terminal marker Dynamin-1. These results suggest that prohormone convertase 5 is active within early compartments of the neuronal regulated secretory pathway and that it is unlikely to be released with its processed substrates. At the regional level, prohormone convertase 5-immunoreactive perikarya were distributed extensively throughout the forebrain. The most numerous and intensely labeled were detected in the olfactory bulb, cerebral cortex, globus pallidus, endopeduncular and subthalamic nuclei, septum, diagonal band of Broca, magnocellular and medial preoptic areas, supraoptic and arcuate nuclei of the hypothalamus, and anterodorsal, laterodorsal, paraventricular and reticular nuclei of the thalamus. Moderate to dense neuronal labeling was also evident in the olfactory tubercle, caudate-putamen, claustrum, bed nucleus of the stria terminalis, substantia innominata, hippocampus, amygdala, and remaining thalamic and hypothalamic nuclei. This widespread distribution suggests that prohormone convertase 5 is involved in the processing of a variety of neuropeptide and/or neurotrophin precursors in mammalian brain.

Animals↗
PubMed2004

Gene expression patterns of pro-opiomelanocortin-processing enzymes PC1 and PC2 during postnatal development of rat corticotrophs.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the anterior pituitary cells of developing rats by a double staining procedure using in situ RT-PCR and an immunofluorescence technique. In the adult, both PC1 mRNA and PC2 mRNA were expressed in corticotrophs, gonadotrophs, thyrotrophs, and mammotrophs. These cells, except for corticotrophs, had previously been considered to be ones in which proprotein processing does not take place, but both PC1 and PC2 may be necessary to process other proteins, such as granin family proteins, having proteolytic cleavage sites and located in secretory granules of the above trophs. In addition, no PC1 or PC2 mRNA was expressed in somatotrophs, which is consistent with the fact that somatotrophs do not contain these granins. In addition, 7B2 mRNA was expressed in these PC2-positive trophs, suggesting that there is a functional relationship between PC2 and 7B2 proteins. We found that alpha-MSH was expressed in the corticotrophs of the postnatal rat and that the number of alpha-MSH-immunopositive corticotrophs decreased as development proceeded. Because the changes in the pattern of POMC processing are considered to depend on the relative expression levels of PC1 and PC2, PC1 and PC2 mRNAs were examined in corticotrophs during postnatal development. We found a decrease in the number of PC2 mRNA-positive cells, which coincided with one in the number of alpha-MSH-immunopositive corticotrophs, as postnatal development proceeded. Our present data demonstrate that the alpha-MSH production varies directly in accordance with the expression of PC2. We also discuss the possible significance of alpha-MSH production during the postnatal period.

Animals↗
PubMed2004

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.

Animals↗
PubMed1994

The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions.

Six mammalian processing enzymes were recently discovered which exhibit significant similarities to both yeast kexin and bacterial subtilisins. These subtilisin/kexin-like convertases were called furin/PACE, PC1/PC3, PC2, PACE4, PC4 and PC5/PC6. The analysis of the mRNA expression of these convertases in rat tissues and cell lines by Northern blot analysis demonstrated a unique pattern for each enzyme. Thus, although furin and PACE4 mRNA (4.4 kb each) exhibit a widespread tissue distribution only furin is ubiquitously expressed. PACE4 exhibits a major 4.4 kb mRNA form, and in some tissues a 3.9 kb form is detected. PC5 mRNA (3.8 kb major) is more restricted in its distribution than PACE4 and furin, and it exhibits the presence of multiple mRNA forms, resulting in variable lengths of the C-terminal Cys-rich domain. In addition, like furin and PACE4, PC5 is expressed in both regulated and constitutively secreting cells. In contrast, PC1 (3 and 5 kb) and PC2 (2.8 and 5 kb) are primarily expressed in tissues and cells containing secretory granules. Multiple mRNA forms are also detected, but as far as is known none affect their open reading frame and only result in a variable length of the 3' non-coding sequence. Finally, PC4 mRNA (2.8 kb major and 1.9 kb minor) is only expressed in testicular germ cells. Biosynthetic analysis of the zymogen activation of PC1 and PC2 and their cleavage specificity following their cellular co-expression with a number of precursors, demonstrated that although pro-PC1 is rapidly activated to PC1 in the endoplasmic reticulum, pro-PC2 conversion into PC2 is rather slow. The cleavage of pro-PC2 into PC2 starts in the trans Golgi network and is regulated by an endogenous endocrine and neural precursor called 7B2. Although the genetic organization of the convertase genes is very similar, they exhibit unique promoter sequences and only furin and PACE4 genes are localized on the same chromosome.

Amino Acid Sequence↗
PubMed2000

Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing.

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin-secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.

Animals↗
PubMed2003

The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2.

The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.

Animals↗
PubMed1996

Implications of the subtilisin/kexin-like precursor convertases in the development and function of nervous tissues.

Furin, PC1, PC2, and PC5 represent mammalian convertases (PCs) found in endocrine, central and peripheral nervous tissues, which cleave a number of precursors at basic residues normally processed in vivo. Typical bonds cleaved by PCs include the pairs Lys-Arg, Arg-Arg and Arg-X-Lys/Arg-Arg. These cleavage sites have been detected following coexpression of each convertase in cell lines together with different precursors as models, including proopiomelanocortin (POMC), proinsulin and proNGF and proBDNF. The presence of PCs and different precursors was revealed by in situ hybridization or immunocytochemistry in cultured AtT-20 cells, in the developing CNS, pituitary, and pancreatic islets. In an experimental model of epilepsy in which epileptiform activities were provoked by kainic acid administration, we observed a similar transient expression of furin and PC1 as compared to that of NGF and BDNF. In conclusion, it is proposed that under different stimuli various precursors are activated by a unique cocktail of convertases, each of which either alone or in combination with others acts to process inactive precursors, and thereby playing an important role in development and in the plasticity of the neuronal system.

Amino Acid Sequence↗
PubMed2004

Obesity and diabetes in transgenic mice expressing proSAAS.

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.

Animals↗
PubMed1993

Human somatostatin receptor genes: localization of SSTR5 to human chromosome 20p11.2.

The gene encoding the somatostatin receptor subtype designated as SSTR5 was mapped to human chromosome 20p11.2 by using fluorescence in situ hybridization to metaphase chromosomes. Fluorescence in situ hybridization using a probe for SSTR5 in combination with probes for neuroendocrine convertase-2 (NEC2), thrombomodulin (THBD), and brain glycogen phosphorylase (PYGB) established a physical order for these loci of 20pter-NEC2-SSTR5-THBD-PYGB-cen.

Chromosome Mapping↗
PubMed2001

Distribution of proSAAS-derived peptides in rat neuroendocrine tissues.

Using a technique to identify substrates of the peptide processing enzyme carboxypeptidase E (CPE), several novel peptides were detected in the brain and pituitary of Cpe(fat)/Cpe(fat) mice and found to be derived from a single precursor, termed proSAAS. In order to gain further information regarding the potential physiological roles of these peptides, we have examined the distribution of two proSAAS-derived peptides, ARPVKEPRSLSAASAPLAETSTPLRL (SAAS) and LENSSPQAPARRLLPP (LEN), in rat neuroendocrine tissues using immunohistochemistry. Both peptides are detected throughout the brain, with the highest concentrations of SAAS peptide in the hypothalamus. In the hippocampus, both peptides are co-localized with prohormone convertase 1 in the dentate gyrus and CA1-3 region. In cerebellum, SAAS peptide is co-localized with prohormone convertase 1 in Purkinje and granular cells, whereas LEN is much more abundant in the Purkinje cells relative to the granular cells. Similarly, SAAS and prohormone convertase 1 are co-localized in the dorsal horn of the spinal cord, while LEN is mainly restricted to fibers of the white matter. In the pituitary, SAAS, LEN, and prohormone convertase 1 are detected in all three lobes. In the pancreas, SAAS, LEN, and prohormone convertase 1 are only detected in the islets, although the peptides are enriched in the peripheral cells (alpha and/or delta) while prohormone convertase 1 is only expressed in the inner cells (beta). Both SAAS and LEN are present in the adrenal medulla along with prohormone convertase 1. Taken together, these data are consistent with the proposed role for proSAAS as an endogenous inhibitor of prohormone convertase 1 in many, but not all cell types. However, the broader localization of the peptides allows for the possibility that they perform additional functions.

Adrenal Glands↗
PubMed2003

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a beta-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3-enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding potential of the isolated P domain, signal peptide-P domain-enhanced GFP constructs with mutated andor truncated P domains were also made. All mutants were expressed in betaTC3 cells, and their subcellular localization and secretion were determined. The mutants fell into three main groups: (i) Golgisecreted, (ii) ERnonsecreted, and (iii) apoptosis inducing. The destabilizing CAT mutations indicate that the side chains of V292, T328, L351, Q408, H409, V412, and F441 and nonpolar fragments of the side chains of R405 and W413 form a hydrophobic patch on CAT that interacts with the P domain. We also have found that the P domain can fold independently, as indicated by its secretion. Interestingly, T594, which is near the P domain C terminus, was not essential for P domain secretion but is crucial for the stability of intact PC3. T594V produced a stable enzyme, but T594D did not, which suggests that T594 participates in important hydrophobic interactions within PC3. These findings support our conclusion that the catalytic and P domains contribute to the folding and thermodynamic stability of the convertases through reciprocal hydrophobic interactions.

Amino Acid Sequence↗
PubMed2004

Modulation of secretory granule-targeting efficiency by cis and trans compounding of sorting signals.

Several protein domains acting through seemingly different mechanisms have been reported to have the capacity to target proteins to dense core secretory granules. Because proteins enter secretory granules with different efficiencies and because some of these proteins contain more than one granule-targeting motif, we have investigated whether compounding sorting signals could alter the efficiency of protein entry into secretory granules. In the current study we demonstrate that a paired basic cleavage site from human prorenin and an alpha-helix-containing secretory granule-sorting signal from the prohormone convertase PC1/3 can synergize to increase granule-sorting efficiency not only when located on the same protein, but also when located on distinct proteins that associate in the secretory pathway.

Animals↗
PubMed2001

Constitutive alpha-secretase cleavage of the beta-amyloid precursor protein in the furin-deficient LoVo cell line: involvement of the pro-hormone convertase 7 and the disintegrin metalloprotease ADAM10.

The beta-amyloid precursor protein (betaAPP) undergoes a physiological cleavage triggered by one or several proteolytic activities referred to as alpha-secretases, leading to the secretion of sAPPalpha. Several lines of evidence indicate that the alpha-secretase cleavage is a highly regulated process. Thus, besides constitutive production of sAPPalpha, several studies have reported on protein kinase C-regulated sAPPalpha secretion. Studies aimed at identifying alpha-secretase(s) candidates suggest the involvement of enzymes belonging to the pro-hormone convertases and disintegrin families. The delineation of respective contributions of proteolytic activities in constitutive and regulated sAPPalpha secretion is rendered difficult by the fact that the overall regulated response always includes the basal constitutive counterpart that cannot be selectively abolished. Here we report on the fact that the furin-deficient LoVo cells are devoid of regulated PKC-dependent sAPPalpha secretion and therefore represent an interesting model to study exclusively the constitutive sAPPalpha secretion. We show here, by a pharmacological approach using selective inhibitors, that pro-hormone convertases and proteases of the ADAM (disintegrin metalloproteases) family participate in the production/secretion of sAPPalphas in LoVo cells. Transfection analysis allowed us to further establish that the pro-hormone convertase 7 and ADAM10 but not ADAM17 (TACE, tumour necrosis factor alpha-converting enzyme) likely contribute to constitutive sAPPalpha secretion by LoVo cells.

Amyloid Precursor Protein Secretases↗
PubMed2004

Significance of prohormone convertase 2, PC2, mediated initial cleavage at the proglucagon interdomain site, Lys70-Arg71, to generate glucagon.

To define the biological significance of the initial cleavage at the proglucagon (PG) interdomain site, K70-R71 downward arrow, we created two interdomain mutants, K70Q-R71Q and R71A. Cotransfection studies in GH4C1 cells show significant amounts of glucagon production by PC2 along with some glicentin, glicentin-related polypeptide-glucagon (GRPP-glucagon) and oxyntomodulin from wild-type PG. In contrast, a larger peptide, PG 33-158, and low amounts of GRPP-glucagon are predominantly generated from interdomain mutants. HPLC analysis shows a 5-fold increase in glucagon production by PC2 from wild-type PG and a corresponding 4-fold lower accumulation and secretion of unprocessed precursor relative to interdomain mutants. PC2 generates significant levels of glucagon from a glicentin (PG 1-69) expression plasmid, whereas PC1/3 produces only modest amounts of oxyntomodulin. Employing a major PG fragment (PG 72-158) expression plasmid, we show that PC1/3 predominantly generates glucagon-like peptide (GLP)-1, whereas PC2 produces only N-terminally extended GLP-1. Surprisingly, production of GLP-1 and GLP-2 by PC1/3 from interdomain mutants, compared with wild-type PG, is not significantly impaired. In addition to PC2 and PC1/3, PC5/6A and furin are also able to cleave the sites, K70-R71 downward arrow and R107-X-R-R110 downward arrow in PG. We show a much greater ability of furin to cleave the monobasic site, R77 downward arrow, than at the dibasic site, R124-R125 downward arrow, which is also weakly processed by PC5/6A, indicating overlapping specificities of these two convertases mainly with PC1/3. We propose here a trimer-like model of the spatial organization of the hormonal sequences within the PG molecule in which the accessibility to prohormone convertase action of most cleavage sites is restricted with the exception of the interdomain site, K70-R71, which is maximally accessible.

Animals↗
PubMed1995

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

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

Amino Acid Sequence↗
PubMed1995

Arg21 is the preferred kexin cleavage site in parathyroid-hormone-related protein.

Parathyroid-hormone-related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post-translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1-141) might be of physiological significance. Cleavage products were identified by N-terminal sequencing. Kex2 preferentially cleaved PTHrP(1-141) carboxy to the triplet arginine site Arg-Arg-Arg21 with a Km of 3.3 +/- 1.7 microM and a kcat of 6 +/- 1.2 s-1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(1-34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys-Arg97, Lys-Arg105, Arg-Arg106 and Thr-Arg108. Cleavage carboxy to Lys-Arg105, the best of these minor sites, occurred with a Km of 8.4 +/- 2.7 microM and a kcat of 0.8 +/- 0.2 s-1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(1-141) is carboxy to Arg-Arg-Arg21, which effectively destroys its parathyroid-hormone-like biological activity. Cleavage of this site by Kex2-related mammalian convertases in vivo may be an important mechanism for full elaboration of the non-parathyroid-hormone-like paracrine actions of PTHrP in a tissue-specific manner.

Arginine↗