Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.     

The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation.

The propeptide of furin has multiple roles in guiding the activation of the endoprotease in vivo. The 83-residue N-terminal propeptide is autoproteolytically excised in the endoplasmic reticulum (ER) at the consensus furin site, -Arg(104)-Thr-Lys-Arg(107)-, but remains bound to furin as a potent autoinhibitor. Furin lacking the propeptide is ER-retained and proteolytically inactive. Co-expression with the propeptide, however, restores trans-Golgi network (TGN) localization and enzyme activity, indicating that the furin propeptide is an intramolecular chaperone. Blocking this step results in localization to the ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network (CGN), suggesting the ER and ERGIC/CGN recognize distinct furin folding intermediates. Following transport to the acidified TGN/endosomal compartments, furin cleaves the bound propeptide at a second, internal P1/P6 Arg site (-Arg-Gly-Val(72)-Thr-Lys-Arg(75)-) resulting in propeptide dissociation and enzyme activation. Cleavage at Arg(75), however, is not required for proper furin trafficking. Kinetic analyses of peptide substrates indicate that the sequential pH-modulated propeptide cleavages result from the differential recognition of these sites by furin. Altering this preference by converting the internal site to a canonical P1/P4 Arg motif (Val(72) --> Arg) caused ER retention and blocked activation of furin, demonstrating that the structure of the furin propeptide mediates folding of the enzyme and directs its pH-regulated, compartment-specific activation in vivo.     

The Mechanism by Which a Propeptide-encoded pH Sensor Regulates Spatiotemporal Activation of Furin

The proprotein convertase furin requires the pH gradient of the secretory pathway to regulate its multistep, compartment-specific autocatalytic activation. Although His-69 within the furin prodomain serves as the pH sensor that detects transport of the propeptide-enzyme complex to the trans-Golgi network, where it promotes cleavage and release of the inhibitory propeptide, a mechanistic understanding of how His-69 protonation mediates furin activation remains unclear. Here we employ biophysical, biochemical, and computational approaches to elucidate the mechanism underlying the pH-dependent activation of furin. Structural analyses and binding experiments comparing the wild-type furin propeptide with a nonprotonatable His-69 → Leu mutant that blocks furin activation in vivo revealed protonation of His-69 reduces both the thermodynamic stability of the propeptide as well as its affinity for furin at pH 6.0. Structural modeling combined with mathematical modeling and molecular dynamic simulations suggested that His-69 does not directly contribute to the propeptide-enzyme interface but, rather, triggers movement of a loop region in the propeptide that modulates access to the cleavage site and, thus, allows for the tight pH regulation of furin activation. Our work establishes a mechanism by which His-69 functions as a pH sensor that regulates compartment-specific furin activation and provides insights into how other convertases and proteases may regulate their precise spatiotemporal activation.     

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.     

Processing and sorting of the prohormone convertase 2 propeptide

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.     

Sequence requirements for precursor cleavage within the constitutive secretory pathway.

We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway. In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells. The results delineate the following sequence rules that govern the constitutive precursor cleavage. (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary. (b) At position -2, a Lys residue is more preferable than Arg. (c) At position -4, an Arg residue is more preferable than Lys. (d) At position 1, a hydrophobic aliphatic amino acid is not suitable.     

Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.     

Positional and additive effects of basic amino acids on processing of precursor proteins within the constitutive secretory pathway

We have recently shown that the Arg/Lys-X-Lys/Arg-Arg or Arg/Lys-X-X-X-Lys/Arg-Arg sequence serves as a signal for cleavage of precursor proteins within the constitutive secretory pathway, and this cleavage is catalyzed by furin, a mammalian homolog of the yeast Kex2 protease. In this study, we further examined sequence requirements for the constitutive precursor cleavage. Based on the data concerning cleavage efficiencies of various prorenin mutants with amino acid substitution(s) around the native cleavage site expressed in CHO cells, we revised the sequence rules that govern the constitutive cleavage as follows: (i) the Arg residue at position −1 is essential; (ii) in addition to the Arg at position −1, at least two out of the three basic residues at positions −2, −4, and −6 are required for efficient cleavage (the presence of all the three basic residues results in most efficient cleavage); (iii) at position +1, a hydrophobic aliphatic amino acid is not suitable.     

A furin-like convertase mediates propeptide cleavage of BACE, the Alzheimer's beta -secretase

The novel transmembrane aspartic protease BACE (for Beta-site APP Cleaving Enzyme) is the beta-secretase that cleaves amyloid precursor protein to initiate beta-amyloid formation. As such, BACE is a prime therapeutic target for the treatment of Alzheimer's disease. BACE, like other aspartic proteases, has a propeptide domain that is removed to form the mature enzyme. BACE propeptide cleavage occurs at the sequence RLPR downward arrowE, a potential furin recognition motif. Here, we explore the role of furin in BACE propeptide domain processing. BACE propeptide cleavage in cells does not appear to be autocatalytic, since an inactive D93A mutant of BACE is still cleaved appropriately. BACE and furin co-localize within the Golgi apparatus, and propeptide cleavage is inhibited by brefeldin A and monensin, drugs that disrupt trafficking through the Golgi. Treatment of cells with the calcium ionophore, leading to inhibition of calcium-dependent proteases including furin, or transfection with the alpha(1)-antitrypsin variant alpha(1)-PDX, a potent furin inhibitor, dramatically reduces cleavage of the BACE propeptide. Moreover, the BACE propeptide is not processed in the furin-deficient LoVo cell line; however, processing is restored upon furin transfection. Finally, in vitro digestion of recombinant soluble BACE with recombinant furin results in complete cleavage only at the established E46 site. Taken together, our results strongly suggest that furin, or a furin-like proprotein convertase, is responsible for cleaving the BACE propeptide domain to form the mature enzyme.     

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.     

Characterization of proADAMTS5 processing by proprotein convertases

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.     

Propeptides Are Sufficient to Regulate Organelle-Specific pH-Dependent Activation of Furin and Proprotein Convertase 1/3

The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles.     

Regulation of ADAMTS9 secretion and enzymatic activity by its propeptide

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.     

The prodomain of a secreted hydrophobic mini-protein facilitates its export from the endoplasmic reticulum by hitchhiking on sorting receptors

Misfolded secretory proteins are retained in the endoplasmic reticulum (ER) by quality control mechanisms targeted to exposed hydrophobic surfaces. Paradoxically, certain conotoxins expose extensive hydrophobic surfaces upon folding to their bioactive structures. How then can such secreted mini-proteins traverse the secretory pathway? Here we show that secretion of the hydrophobic conotoxin-TxVI is strongly dependent on its propeptide domain, which enhances TxVI export from the ER. The propeptide domain interacts with sorting receptors from the sortilin Vps10p domain family. The sortilin-TxVI interaction occurs in the ER, and sortilin facilitates export of TxVI from the ER to the Golgi. Thus, the prodomain in a secreted hydrophobic protein acts as a tag that can facilitate its ER export by a hitchhiking mechanism.     

Maturation of the trans-Golgi network protease furin: compartmentalization of propeptide removal,substrate cleavage,and COOH-terminal truncation

We have cloned a bovine cDNA encoding the trans-Golgi network (TGN) protease furin and expressed it via recombinant vaccinia viruses to investigate intracellular maturation. Pulse-chase labeling reveals that the 104-kD pro-furin bearing high mannose N-glycans is rapidly processed into the 98-kD protease whose N-glycans remain sensitive to endoglycosidase H for a certain period of time. Furthermore, in the presence of brefeldin A, pro-furin cleavage occurs. From these data we conclude that the ER is the compartment of propeptide removal. Studies employing the ionophore A23187 and DTT show that autocatalysis is Ca2+ dependent and that it does not occur under reducing conditions. Pro- furin produced under these conditions never gains endo H resistance indicating that it is retained in the ER. Coexpression of furin with the fowl plague virus hemagglutinin in the presence of brefeldin A and monensin reveals that furin has to enter the Golgi region to gain substrate cleaving activity. N-glycans of furin are sialylated proving its transit through the trans-Golgi network. A truncated form of furin is found in supernatants of cells. Truncation is inhibited in the absence of Ca2+ ions and in the presence of acidotropic agents indicating that it takes place in an acidic compartment of cells. Comparative analysis with furin expressed from cDNA reveals that the truncated form prevails in preparations of biologically active, endogenous furin obtained from MDBK cells. This observation supports the concept that secretion of truncated furin is a physiological event that may have important implications for the processing of extracellular substrates.     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.     

Differential modulation of prM cleavage, extracellular particle distribution, and virus infectivity by conserved residues at nonfurin consensus positions of the dengue virus pr-M junction

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.     

Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus

The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.     

The Furin Protease Cleavage Recognition Sequence of Sindbis Virus PE2 Can Mediate Virion Attachment to Cell Surface Heparan Sulfate

Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.     

A transient forward-targeting element for microneme-regulated secretion in Toxoplasma gondii.

BACKGROUND INFORMATION: Accurate sorting of proteins to the three types of secretory granules in Toxoplasma gondii is crucial for successful cell invasion by this obligate intracellular parasite. As in other eukaryotic systems, propeptide sequences are a common yet poorly understood feature of proteins destined for regulated secretion, which for Toxoplasma occurs through two distinct invasion organelles, rhoptries and micronemes. Microneme discharge during parasite apical attachment plays a pivotal role in cell invasion by delivering adhesive proteins for host receptor engagement. RESULTS: We show here that the small micronemal proprotein MIC5 (microneme protein-5) undergoes proteolytic maturation at a site beyond the Golgi, and only the processed form of MIC5 is secreted via the micronemes. Proper cleavage of the MIC5 propeptide relies on an arginine residue in the P1' position, although P1' mutants are still cleaved to a lesser extent at an alternative site downstream of the primary site. Nonetheless, this aberrantly cleaved species still correctly traffics to the micronemes, indicating that correct cleavage is not necessary for micronemal targeting. In contrast, a deletion mutant lacking the propeptide was retained within the secretory system, principally in the ER (endoplasmic reticulum). The MIC5 propeptide also supported correct trafficking when exchanged for the M2AP propeptide, which was recently shown to also be required for micronemal trafficking of the TgMIC2 (T. gondii MIC2)-M2AP complex [Harper, Huynh, Coppens, Parussini, Moreno and Carruthers (2006) Mol. Biol. Cell 17, 4551-4563]. CONCLUSION: Our results illuminate common and unique features of micronemal propeptides in their role as trafficking facilitators.