Spatial and temporal expression of fibril-forming minor collagen genes (types V and XI) during fracture healing

Skeletal development involves the coordinated participation of several types of collagen, including both major and minor fibrillar collagens. Although much is known about the major fibrillar collagens, such as types I and II, less is known about the minor fibrillar collagens, and their role in the repair and regeneration of bone has not been extensively studied. To clarify the role of minor fibrillar collagens in fracture repair, we examined the spatial and temporal expression of mRNAs for pro-alpha 2(V) collagen and pro-alpha 1(XI) collagen in healing fractures in the rat by in situ hybridization and compared their patterns of expression with those of mRNAs for pro-alpha 1(I) collagen, pro-alpha 1(II) collagen, and osteocalcin. A strong signal for pro-alpha 2(V) was detected in the periosteal osteoprogenitor cells, whereas osteocalcin mRNA was strongly expressed only in the deep layers of the hard callus. The distribution of the pro-alpha 2(V) signal was correlated with that of pro-alpha 1(I) but was mutually exclusive of that of pro-alpha 1(II). The expression of pro-alpha 1(XI) mRNA was synchronously regulated with that of pro-alpha 1(II) during chondrogenesis in the soft callus. In the hard callus, pro-alpha 1(XI) signal was found in osteoblastic cells at the site of intramembranous and endochondral ossification. These cells simultaneously expressed pro-alpha 2(V), although they were negative for pro-alpha 1(II). These findings suggest that the alpha 2(V) collagen chain participates in the formation of the noncartilaginous fibrillar network in the hard callus and preferentially contributes to the initial stage of the intramembranous bone formation. Recent reports have revealed that type-XI collagen, which had been classified as a cartilage-type collagen, is not necessarily specific for cartilage. The present results advanced this recognition and demonstrated a coexpression of alpha 1(XI) mRNA and alpha 2(V) mRNA in the noncartilaginous tissues in the fracture callus; this suggests the presence of tissue-specific and stage-specific heterotrimers consisting of alpha 1(XI) and alpha 2(V) collagen chains and the association of such hybrid trimers with the major fibrillar collagens in the process of fracture healing.     

Differential cleavage of provasopressin by the major molecular forms of SPC3

We have investigated the roles of full-length and carboxyl-terminus-truncated forms of the subtilisin-like prohormone convertase SPC3 in the processing of the radiolabeled vasopressin and oxytocin precursors, in vitro. We found SPC3 cleaves provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide processing sites. Prooxytocin is cleaved by SPC3 at the oxytocin-neurophysin cleavage site. However, our results reveal differences in processing of provasopressin by the different molecular forms of SPC3. In incubations where the rate of autocatalytic carboxyl-terminus truncation of SPC3 was dramatically reduced, 86-kDa SPC3, which has an unprocessed carboxyl terminus, cleaved provasopressin at the neurophysin-glycopeptide junction. Cleavage at the vasopressin-neurophysin junction only occurred with the appearance of carboxyl-terminus-truncated forms of the enzyme. Incubations containing 64-kDa SPC3 or 64-kDa SPC3-T, a recombinant form of SPC3 truncated 14 amino acids beyond the conserved carboxyl-terminal "P-domain," rapidly cleaved provasopressin at both the vasopressin-neurophysin and neurophysin-glycopeptide junctions. Our results also suggest that prooxytocin is unable to be cleaved by the 86-kDa form of SPC3. We propose that SPC3 should be considered as a candidate endoprotease in the biosynthesis of vasopressin. Furthermore, we suggest that the carboxyl terminus of SPC3 alters the cleavage specificity of SPC3.     

In vitro cleavage of internally quenched fluorogenic human proparathyroid hormone and proparathyroid-related peptide substrates by furin. Generation of a potent inhibitor

The cleavage of parathyroid hormone (PTH) from its precursor proparathyroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazure, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525). We also showed that a synthetic peptide comprising the -6 to +7 sequence of human pro-PTH is appropriately cleaved by purified furin in vitro. The human pro-PTH processing site Lys-Ser-Val-Lys-Lys-Arg differs from the consensus furin site Arg-Xaa-(Lys/Arg)-Arg that is represented by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related peptide (pro-PTHrP). An earlier study demonstrated that an internally quenched fluorogenic substrate bearing an O-aminobenzoyl fluorescent donor at the NH2 terminus and an acceptor 3-nitrotyrosine near the COOH terminus was appropriately cleaved by the convertases furin and PC1 (Jean, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Biochem. J. 307, 689-695). Here, we have synthesized a series of internally quenched fluorogenic substrates based upon the pro-PTH and pro-PTHrP sequences to determine which residues are important for furin cleavage. Purified recombinant furin and PC1 cleaved the human pro-PTH internally quenched substrate at the appropriate site in an identical manner to that observed with the nonfluorescent peptide. Several substitutions in the P6-P3 sequence were well tolerated; however, replacement of the Lys at the P6 position with Gly and replacement of the P3 Lys by an acidic residue led to markedly compromised cleavage by furin. Furin activity was very sensitive to substitution in P' positions. Replacement of Ser at P1' with Gly and Val at P2' with Ala generated substrates that were less well cleaved. Substitution at the P1' position of Val for Ser in conjunction with Ala for Val at P2', as well as a single substitution of Lys for Val at P2', generated specific inhibitors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.     

Evaluation in clinical practice: problems, precedents and principles

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated. Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA. Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the -1 and -2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions -1 and either -2 or -4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.     

Human prochymase activation. A novel role for heparin in zymogen processing

Human prochymase is packaged with heparin in mast cell granules and appears to be activated by dipeptidylpeptidase I. We show that a high affinity interaction between heparin and prochymase allows the 2-residue propeptide to be cleaved by dipeptidylpeptidase I. A conserved Glu in the propeptide is necessary for this heparin effect. Following propeptide cleavage, capture of the newly generated NH2 terminus by an "activation groove" on the enzyme activates the enzyme and concurrently prevents a progressive degradation of the NH2 terminus by dipeptidylpeptidase I. Surrogate peptide studies show that the activation groove is unoccupied in prochymase and is specific for the chymase NH2 terminus. These observations indicate that heparin is an important cofactor in the prochymase activation process and explain how dipeptidylpeptidase I, a nonspecific processing enzyme, can effect a specific cleavage of the zymogen propeptide.     

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development

Bone morphogenetic protein-4 (BMP-4) is a multifunctional developmental regulator. BMP-4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif -Arg-Ser-Lys-Arg-. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif -Arg-X-Arg/Lys-Arg-. To examine potential roles of PCs during embryonic development we have misexpressed a potent protein inhibitor of furin, alpha1-antitrypsin Portland (alpha1-PDX) in early Xenopus embryos. Ectopic expression of alpha1-PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. alpha1-PDX-mediated neural induction can be reversed by co-expression of downstream components of the BMP-4 signaling pathway. Thus, alpha1-PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP-4 convertase(s). Consistent with this hypothesis, alpha1-PDX prevents cleavage of BMP-4 in an oocyte translation assay. Using an in vitro digestion assay, we demonstrate that four members of the PC family have the ability to cleave BMP-4, but of these, only furin and PC6B are sensitive to alpha1-PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP-4 during vertebrate embryogenesis.     

Endoproteolysis at tetrabasic amino acid sites in procalcitonin gene-related peptide by pituitary cell lines

The specificity of neuroendocrine prohormone convertases for tetrabasic amino acid sites was investigated. Mutations were introduced into the tetrabasic cleavage site of the procalcitonin gene-related peptide (proCGRP) cDNA and these mutated cDNA's were expressed in AtT-20 cells which predominantly express the endoprotease prohormone convertase-1 (PC1/3), and in GH3 cells which predominantly express prohormone convertase-2 (PC2). Mutations were introduced into the proCGRP cDNA which converted the wild-type ArgArgArgArg site to LysLysArgArg and ArgArgLysLys, and the proCGRP variants were stably transfected into AtT-20 and GH3 cells. ProCGRP containing each of the LysLysArgArg permutations were efficiently cleaved in both AtT-20 and GH3 cells. Cleavage of LysLysArgArg in exogenous proCGRP, but not in endogenous POMC, suggests that the specificity of cleavage at tetrabasic sites is not defined solely by the endoproteases expressed by the cell or by the amino acid sequence at the cleavage site, but is also dependent on the structure of the propeptide.     

Proanthocyanidin-rich extract from grape seeds attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.     

Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence

Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form. The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured. The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH. The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity. Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin.     

Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases

Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.     

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.     

Zinc-dependent dimers observed in crystals of human endostatin

The crystal structure of human endostatin reveals a zinc-binding site. Atomic absorption spectroscopy indicates that zinc is a constituent of both human and murine endostatin in solution. The human endostatin zinc site is formed by three histidines at the N terminus, residues 1, 3, and, 11, and an aspartic acid at residue 76. The N-terminal loop ordered around the zinc makes a dimeric contact in human endostatin crystals. The location of the zinc site at the amino terminus, immediately adjacent to the precursor cleavage site, suggests the possibility that the zinc may be involved in activation of the antiangiogenic activity following cleavage from the inactive collagen XVIII precursor or in the cleavage process itself.     

Mouse hepatitis virus 3C-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro

The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.     

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.     

Processing of the Borna disease virus glycoprotein gp94 by the subtilisin-like endoprotease furin

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.     

Mapping of the complement regulatory domains in the human factor H-like protein 1 and in factor H1

The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

Expression of influenza B virus hemagglutinin containing multibasic residue cleavage sites

The hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA0 precursor is not cleaved to the HA1 and HA2 subunits by tissue culture cell-associated proteases. To investigate if an HA protein could be obtained that could be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. Three HA mutant proteins were constructed, through substitution or insertion of arginine residues, that have 4, 5, or 6 basic residues at their cleavage sites. Chemical cross-linking studies indicated that all three HA cleavage site mutants could oligomerize to a trimeric species, like WT HA. The three HA cleavage site mutant proteins were efficiently transported to the cell surface and bound erythrocytes in hemadsorption assays. The mutants were cleaved at a low level to HA1 and HA2 by an endogenous host cell protease and cleavage could be increased somewhat by addition of exogenous trypsin. The fusogenic activities of the HA cleavage site mutants were assessed in comparison to the WT HA protein by determining their syncytium formation ability and by using an R18 lipid-mixing assay and a NBD-taurine aqueous-content mixing assay. While the fusion activity of the WT HA protein was dependent on exogenous trypsin to activate HA, the three HA cleavage site mutant proteins were able to induce fusion in the absence of trypsin when assayed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing assays, but were unable to induce syncytium formation in either the presence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the influenza B virus HA1/HA2 boundary does enable some HA0 molecules to be cleaved intracellularly, it alone is not sufficient for efficient cleavage.