Fluorogenic peptides encompassing the processing sites of envelope glycoproteins of the infectious influenza A Hong Kong virus (HKV), Ebola virus (EBOV) and respiratory syncytial virus (RSV) were tested for cleavage by soluble recombinants of the proprotein convertases furin, PC5 and PC7. Kinetic studies with these intramolecularly quenched fluorogenic peptides revealed selective cleavages at the physiological dibasic sites. The HKV peptide is cleaved by both furin and PC5 with similar efficacy; in comparison, PC7 cleaves this substrate poorly. In contrast with the basic tetrapeptide insertion within the haemagglutinin sequence of HKV, two other dipeptide insertions revealed a poorer cleavage with a similar rank order of potency. These results demonstrate that the N-terminal RERR insertion to the wild-type avian RKKR↓ sequence is functionally significant, and suggest that the approx. 5-fold increase in cleavage efficacy contributes to the high infectivity of the H5N1 virus subtype. With regard to RSV peptide processing, PC7 is twice as effective as PC5 and furin. The EBOV peptide was processed with similar efficiency by the three enzymes. Our observations that all of these cleavages can be effectively inhibited by a plant andrographolide derivative at 250µM or less might aid in the design of potent convertase inhibitors as alternative antiviral therapies.

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca2+-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK137- andRRLL186-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P′ residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100–200 nm.

‡ Biochemical Neuroendocrinology, Clinical Research Institute of Montréal, 110 Pine Ave West, Montreal, QC, Canada H2W 1R7 ¶ Diseases of Aging Unit, Loeb Health Research Institute at the Ottawa Hospital, 725 Parkdale Ave, Ottawa, ON K1Y 4K9 Canada ** Neuropeptides Structures and Metabolism, Clinical Research Institute of Montréal, 110 Pine Ave West, Montreal, QC H2W 1R7 Canada || University of Chicago, Department of Pharmacological and Physiological Sciences, 947 East, 58th Street, Chicago, IL 60637 USA § IPMC du CNRS, UPR411, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France ‡‡ To whom correspondence and reprint requests should be addressed.

Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.

ABSTRACT Mouse mammary tumor viruses express a superantigen essential for their life cycle. It has been proposed that viral superantigens (vSags) require processing by prohormone convertases (PCs) for activity. We now observe, using a panel of mutant forms of potential PC cleavage sites and in vitro cleavage assays, that only the CS1 (position 68 to 71) and CS2 (position 169 to 172) sites are utilized by furin and PC5. Other members of the convertase family that are expressed in lymphocytes are not endowed with this activity. Furthermore, mutant forms of two different viral superantigens, vSag7 and vSag9, which completely abrogated in vitro processing by convertases, were efficient in functional presentation to responsive T-cell hybridomas. This effect was observed in both endogenous presentation and paracrine transfer of the vSag. Processing by convertases thus appears not to be essential for vSag function. Finally, we have identified the purified endosomal protease cathepsin L as another protease that is able to cleave convertase mutant vSag in vitro, yielding fragments similar to those detected in vivo, thus suggesting that proteases other than convertases are involved in the activation of vSags.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

The prohormone convertase PC2 is one of the major subtilisin/kexin‐like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin‐like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2‐specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2‐specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site‐directed mutagenesis of Tyr 194 and of the oxyanion hole Asp 309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into β‐endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D‐PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co‐immunoprecipitations and Western blots, binding assays indicate that Tyr 194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp 309 is critical for the binding of proPC2 with pro7B2.

A 32 amino acid peptide called histatin‐3 (H3; 22% His) and its TV‐terminal 24 amino acid fragment histatin‐5 (H5, 33% His), are found in human saliva and possess powerful antimicrobial properties. These His‐rich peptides have been synthesized by Fmoc‐based solid‐phase chemistry. Their sequences are: DSHAKRHHGYKRKFHEKHHSHRGYRSNYLYDN (H3) and DSHAKRHHGYKRKFHEKHHSHRGY (H5). In addition, we also prepared two H5 and one H3 mutants. The H5 mutants were: DH5 (all amino acids in D configuration) and H5F (where all His are replaced by Phe at positions 3, 7, 8, 15, 18, 19, 21). The 9–24 segment of H3 with all the His at positions 15, 18, 19, 21 replaced by Tyr was also prepared (Δ1–8H3Y). The behavior of these five peptides was examined with three proprotein convertases (PC's) which possess cleavage specificity directed towards single and pairs of basic residues. These were: human (h)PCl, an endocrine and neural convertase, hfurin and rat (r)PC7, two widely expressed enzymes. All are serine endoproteases belonging to the kexin/subtilisin family. Our in vitro study revealed that H3 behaves as a substrate for PCI, being cleaved by this endoprotease primarily at a site carboxy terminal to the single Arg25 residue (HRGYR↓SN). On prolonged incubation some minor cleavage was also observed C‐terminal to the first Lys Arg6 pairs of basic amino acids namely at: HAKR↓HH, which contains a P4 as well as P'l and P'2 His residues. The second potential site YKRK12‐FH which does not have a P4 basic residues is not cleaved, even upon incubation with excess protease. PCI only poorly cleaves H5 at the same site mentioned above for H3, i.e., at HAKR↓HH. As expected, neither the D‐amino acid analogue (DH5), nor the Phe and Tyr mutant analogues of the long and short histatins, respectively, are cleaved at all.In contrast to the above findings for hPCl, the convertase hfurin did not cleave any of the five synthetic peptides. Instead, H3 and H5 were found to be moderately potent inhibitors of the furin‐mediated cleavage of the pentapeptide pGlu‐Arg‐Thr‐Lys‐Arg‐MCA fluorogenic substrate. This inhibition was reversible and competitive, with an estimated inhibition constant Ki of 1.98 μM for H3 and 2.98 μM for H5. The other analogs exhibited only a moderate to weak inhibition of furin, suggesting that substitution of all His by aromatic residues (Phe or Tyr) drastically reduces their inhibitory potency. When tested against rPC7, H3 exhibited almost identical inhibition profile with a measured Ki of 2.4 μM. The partial sequence identity of H3 to the inhibitory pro‐peptide of furin and PC7 provides a rationale for our observation. © Munksgaard 1997.

We studied the extent of cellular inhibitory activity of alpha 1-antitrypsin Portland (alpha 1-PDX), a patent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha 1-PDX on the intracellular processing of two model precursors (pro ?Ba and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PCB-A, PC5-B, and PC7, The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha 1-PDX in AtT20 cells. Results revealed that alpha 1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha 1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha 1-PDX (64 kDa), the cleaved (56 kDa) secreted product; does not significantly inhibit furin activity in vitro, Cellular expression of alpha 1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha 1-PDX cells demonstrated that alpha 1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

The objective of this study has been to delineate the side-specific effects of Na+ and K+ on the transport kinetics of tissue-specific Na/K pumps. Two experimental systems have been used. In one, Na/K pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E.A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925-16930), with the alpha 2 and alpha 3 isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the alpha 1 catalytic isoform, the axolemma microsomes were predominantly alpha 3 (approximately 60%) with lesser amounts of alpha 2 (approximately 25%) and alpha 1 (approximately 15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (alpha 1 of kidney, alpha 1 and alpha 2 of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na+ and extracellular K+ on pump-mediated 86Rb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the alpha 3 isoform compared with the alpha 1 and alpha 2 isoforms were observed, i.e. a approximately 3-fold higher affinity for extracellular K+ and approximately 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 mM extracellular Na+, apparent K0.5 values for extracellular K+ activation of K+(Rb+) influxes were 0.22 +/- 0.02 mM for alpha 1, 0.20 +/- 0.02 mM for alpha 2, and 0.09 +/- 0.01 mM for alpha 3. At high intracellular K+ (> or = 100 mM) and saturating extracellular K+ concentrations, apparent K0.5 values for cytoplasmic Na+ activation were 17.6 +/- 1.1 mM for alpha 1, 19.7 +/- 1.0 mM for alpha 2, and 63.5 +/- 9.1 mM for alpha 3. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. Axolemma pumps had a approximately 3-fold lower K0.5 for extracellular K+ and a approximately 2-fold higher K0.5 for cytoplasmic Na+.(ABSTRACT TRUNCATED AT 400 WORDS)

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low uncoupled Na+/O flux activity.