Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Diminished prohormone convertase 3 expression (PC1/PC3) inhibits progastrin post-translational processing

Gastrin is initially synthesized as a large precursor that requires endoproteolytic cleavage by a prohormone convertase (PC) for bioactivation. Gastric antral G-cells process progastrin at Arg(94)Arg(95) and Lys(74)Lys(75) residues generating gastrin heptadecapeptide (G17-NH(2)). Conversely, duodenal G-cells process progastrin to gastrin tetratriacontapeptide (G34-NH(2)) with little processing at Lys(74)Lys(75). Both tissues express PC1/PC3 and PC2. Previously, we demonstrated that heterologous expression of progastrin in an endocrine cell line that expresses PC1/PC3 and little PC2 (AtT-20) resulted in the formation of G34-NH(2). To confirm that PC1/PC3 was responsible for progastrin processing in AtT-20 cells and capable of processing progastrin in vivo we coexpressed either human wild-type (Lys(74)Lys(75)) or mutant (Arg(74)Arg(75), Lys(74)Arg(75), and Arg(74)Lys(75)) progastrins in AtT-20 cells with two different antisense PC1/PC3 constructs. Coexpression of either antisense construct resulted in a consistent decrease in G34-NH(2) formation. Gastrin mRNA expression and progastrin synthesis were equivalent in each cell line. Although mutation of the Lys(74)Lys(75) site within G34-NH(2) to Lys(74)Arg(75) resulted in the production of primarily G17-NH(2) rather than G34-NH(2), inhibition of PC1/PC3 did not significantly inhibit processing at the Lys(74)Arg(75) site. We conclude that PC1/PC3 is a progastrin processing enzyme, suggesting a role for PC1/PC3 progastrin processing in G-cells.     

Proteolytic processing of chromogranin A by the prohormone convertase PC2

The neuroendocrine secretory protein chromogranin A (CgA) is a precursor for various biologically active peptides. Several single and paired basic residues are present within its primary amino acid sequence comprising cleavage sites for prohormone convertases. In this study, SH-SY5Y human neuroblastoma cells were stably transfected with the prohormone convertase PC2 to analyse the proteolytic processing of endogenous chromogranin A and, in particular, the formation of the chromogranin-A-derived peptide GE-25. Our analyses revealed a significant change in the pattern of proteolytic conversion of chromogranin A in cells expressing PC2. Mock-transfected control cells contained mainly the intact chromogranin A molecule and hardly any shorter products were found. On the other hand, PC2-transfected cells showed extensive processing of chromogranin A, resulting in significantly lower amounts of the intact precursor and especially high levels of the free peptide GE-25.     

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)     

Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells.

A new family of mammalian subtilisin-like enzymes, probably involved in the processing of proproteins in regulated and constitutive cells at paired basic residues, has recently been discovered. Little information exists as yet concerning the biosynthesis of these endogenous subtilisin-like enzymes. In the present work the biosynthesis and release of the endogenous prohormone convertase PC1 in AtT-20 cells were studied. As predicted from mRNA studies, AtT-20 cells contain high levels of PC1 protein. Through immunoblotting, 87-kilodalton (kDa) and 66-kDa bands were detected with an amino terminally directed antiserum; however, only the 87-kDa product was detected with carboxyl terminally directed antiserum, indicating carboxyl terminal truncation. Pulse-chase experiments, using [35S]methionine/cysteine, showed that after 20 min pulse the main product in the cells was the 87-kDa protein. Cells chased for varying amounts of time exhibited a progressive increase in the intensity of a 66-kDa band, along with a corresponding decrease of the 87-kDa band. The 87-66 kDa conversion was nearly complete after 4 h of chase. This posttranslational processing was inhibited by the ionophore monensin, a Golgi disruptor, with a corresponding accumulation of the 87-kDa protein within the cell. Both the 87 kDa- and 66 kDa-labeled proteins were detected as membrane-bound rather than soluble proteins. The 87-kDa protein was the main product secreted by nonstimulated AtT-20 cells, while the 66-kDa product was only released when the cells were stimulated with CRF or BaCl2 and Bromo-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Cloning and functional expression of a novel endoprotease involved in prohormone processing at dibasic sites.

We cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites. This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2. Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells. The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells. We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells.     

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.     

Activation of C1r by proteolytic cleavage.

C1r was unable to cleave and activate proenzyme C1s unless first incubated at 37 degrees C in the absence of calcium before the addition of C1s. The acquisition of ability to activate C1s was associated with, and paralleled by, cleavage of each of the two noncovalently bonded 95,000 dalton chains of the molecule into disulfide linked subunits of 60,000 and 35,000 daltons, respectively. Thus, C1r is converted from an inactive form into an enzyme, C1r, able to cleave and activate C1s by proteolytic cleavage in marked analogy to the activation of several other complement enzymes. Trypsin was also found to cleave C1r but at a different site, and its action did not lead to C1r activation. C1r activation was inhibited by calcium, polyanethol sulfonate, C1 inactivator, and DFP but not by a battery of other protease inhibitors. C1 inactivator inhibited C1r by forming a complex with C1r via sites located on the light chain of the molecule. In other studies, cleavage of C1r was not accelerated by the addition of C1r ot C1s. C1r and C1r were found to have the same m.w., sedimentation coefficient, and diffusion coefficients. They differed, however, in charge with C1r migrating as a Beta-globulin and C1r as a gammaglobulin on electrophoresis in agarose. The amino acid composition of C1r and of each of the two polypeptide chains of Clr was determined. Both chains contained carbohydrate. Proteolytic cleavage of the C1r molecule was found to occur on addition of aggregated IgG to a mixture of C1q, C1r, and C1s in the presence of calcium. Neither C1q, C1s nor aggregated IgG alone, not C1r nor C1s induced C1r cleavage. Liquoid, an inhibitor of C1 activation, inhibited C1r cleavage. Thus, proteolytic cleavage of C1r appears to be a biologically meaningful event occurring during the activation of C1.     

The role of dibasic residues in prohormone sorting to the regulated secretory pathway. A study with proneurotensin

The mechanisms by which prohormone precursors are sorted to the regulated secretory pathway in neuroendocrine cells remain poorly understood. Here, we investigated the presence of sorting signal(s) in proneurotensin/neuromedin N. The precursor sequence starts with a long N-terminal domain followed by a Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin)-Lys-Arg- sequence and a short C-terminal tail. An additional Arg-Arg dibasic is contained within the neurotensin sequence. Mutated precursors were expressed in endocrine insulinoma cells and analyzed for their regulated secretion. Deletion mutants revealed that the N-terminal domain and the Lys-Arg-(C-terminal tail) sequence were not critical for precursor sorting to secretory granules. In contrast, the Lys-Arg-(neuromedin N)-Lys-Arg-(neurotensin) sequence contained essential sorting information. Point mutation of all three dibasic sites within this sequence abolished regulated secretion. However, keeping intact any one of the three dibasic sequences was sufficient to maintain regulated secretion. Finally, fusing the dibasic-containing C-terminal domain of the precursor to the C terminus of beta-lactamase, a bacterial enzyme that is constitutively secreted when expressed in neuroendocrine cells, resulted in efficient sorting of the fusion protein to secretory granules in insulinoma cells. We conclude that dibasic motifs within the neuropeptide domain of proneurotensin/neuromedin N constitute a necessary and sufficient signal for sorting proteins to the regulated secretory pathway.     

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.     

Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice

Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). In this report we have examined the biosynthesis and processing of proglucagon in isolated islets from these mice via pulse-chase labeling and find that proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.     

Biosynthesis of proTRH-derived peptides in prohormone convertase 1 and 2 knockout mice

Prohormone convertases (PCs) 1 and 2 are the primary endoproteases involved in the post-translational processing of proThyrotropin Releasing Hormone (proTRH) to give rise to TRH and other proposed biologically active non-TRH peptides. Previous evidence suggests that PC1 is responsible for most proTRH cleavage events. Here, we used the PC1 and PC2 knockout (KO) mouse models to examine the effects of PC1 or PC2 loss on proTRH processing. The PC1KO mouse presented a decrease in five proTRH-derived peptides, whereas the PC2KO mouse showed only lesser reduction in three TRH (Gln-His-Pro), TRH-Gly (Gln-His-Pro-Gly), and the short forms preproTRH(178-184) (pFQ(7)) and preproTRH(186-199) (pSE(14)) of pFE(22) (preproTRH(178-199)). Also, PC1KO and not PC2KO showed a decrease in pEH(24) indicating that PC1 is more important in generating this peptide in the mouse, which differs from previous studies using rat proTRH. Furthermore, downstream effects on thyroid hormone levels were evident in PC1KO mice, but not PC2KO mice suggesting that PC1 plays the more critical role in producing bioactive hypophysiotropic TRH. Yet loss of PC1 did not abolish TRH entirely indicating a complementary action for both enzymes in the normal processing of proTRH. We also show that PC2 alone is responsible for catalyzing the conversion of pFE(22) to pFQ(7) and pSE(14), all peptides implicated in regulation of suckling-induced prolactin release. Collectively, results characterize the specific roles of PC1 and PC2 in proTRH processing in vivo.     

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.     

Interaction of Drosophila melanogaster prohormone convertase 2 and 7B2. Insect cell-specific processing and secretion

The prohormone convertases (PCs) are an evolutionarily ancient group of proteases required for the maturation of neuropeptide and peptide hormone precursors. In Drosophila melanogaster, the homolog of prohormone convertase 2, dPC2 (amontillado), is required for normal hatching behavior, and immunoblotting data indicate that flies express 80- and 75-kDa forms of this protein. Because mouse PC2 (mPC2) requires 7B2, a helper protein for productive maturation, we searched the fly data base for the 7B2 signature motif PPNPCP and identified an expressed sequence tag clone encoding the entire open reading frame for this protein. dPC2 and d7B2 cDNAs were subcloned into expression vectors for transfection into HEK-293 cells; mPC2 and rat 7B2 were used as controls. Although active mPC2 was detected in medium in the presence of either d7B2 or r7B2, dPC2 showed no proteolytic activity upon coexpression of either d7B2 or r7B2. Labeling experiments showed that dPC2 was synthesized but not secreted from HEK-293 cells. However, when dPC2 and either d7B2 or r7B2 were coexpressed in Drosophila S2 cells, abundant immunoreactive dPC2 was secreted into the medium, coincident with the appearance of PC2 activity. Expression and secretion of dPC2 enzyme activity thus appears to require insect cell-specific posttranslational processing events. The significant differences in the cell biology of the insect and mammalian enzymes, with 7B2 absolutely required for secretion of dPC2 and zymogen conversion occurring intracellularly in the case of dPC2 but not mPC2, support the idea that the Drosophila enzyme has specific requirements for maturation and secretion that can be met only in insect cells.