©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing Specificity and Biosynthesis of the Drosophila melanogaster Convertases dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2 (*)

(Received for publication, August 30, 1994; and in revised form, October 24, 1994)

Isabelle De Bie (1) Diane Savaria (1) Anton J. M. Roebroek (3) Robert Day (1) Claude Lazure (2) Wim J. M. Van de Ven (3) Nabil G. Seidah (1)(§)

From the  (1)J. A. DeSève Laboratories of Biochemical Neuroendocrinology and (2)Neuropeptides Structure and Metabolism, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada and the (3)Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pro-protein and pro-hormone convertases are subtilisin/kexin-like enzymes implicated in the activation of numerous precursors by cleavage at sites mostly composed of pairs of basic amino acids. Six members of this family of enzymes have been identified in mammals and named furin (also called PACE), PC1 (also called PC3), PC2, PACE4, PC4, and PC5 (also called PC6). Multiple transcripts are produced for all the mammalian convertases, but only in the cases of PC4, PACE4, and PC5 does differential splicing result in the modification of the C-terminal sequence of these enzymes. A similar molecular diversity is also observed for the convertases of Hydra vulgaris, Caenorhabditis elegans, and Drosophila melanogaster. In the third species, two genes homologous to human furin called Dfur1 and Dfur2 have been identified. The Dfur1 gene undergoes differential splicing to generate three type I membrane-bound proteins called dfurin1, dfurin1-CRR, and dfurin1-X, which differ only in their C-terminal sequence. By using recombinant vaccinia viruses that express each of the dfurin proteins, we investigated the potential effect of the C-terminal domain on their catalytic specificities. For this purpose, these enzymes were coexpressed with the precursors pro-7B2, pro-opiomelanocortin, and pro-dynorphin in a number of cell lines, and the processed products obtained were characterized. Our studies demonstrate that these proteases display cleavage specificities similar to that of mammalian furin but not to that of PC2. In contrast, we noted significant differences in the biosynthetic fates of these convertases. All dfurins undergo rapid removal of their transmembrane domain within the endoplasmic reticulum, resulting in the release of several truncated soluble forms. However, in the media of cells containing secretory granules, such as GH4C1 and AtT-20, dfurin1-CRR and dfurin2 predominate over dfurin1, whereas dfurin1-X is never detected. While pro-segment removal occurs predominantly in the trans-Golgi network for all the dfurins, in the presence of brefeldin A, only dfurin1-CRR and dfurin2 can undergo partial zymogen cleavage. The conclusions drawn from the results of this study may well be applicable to the mammalian convertases PC4, PACE4, and PC5, which also display C-terminal sequence heterogeneity.


INTRODUCTION

Post-translational endoproteolysis of precursor proteins is one of the mechanisms by which cells increase the diversity of their biologically active products. Initial cleavage of pro-proteins usually occurs at well defined sites consisting generally of pairs of basic amino acids, frequently Lys-Arg or Arg-Arg, but also at specific monobasic sites usually occupied by a single Arg(1, 2, 3) . Recently, a number of mammalian genes and cDNAs encoding subtilisin-like enzymes have been identified, and these candidate processing enzymes were proposed to be responsible for the cleavage at dibasic and monobasic sites of precursor proteins (for reviews see (3, 4, 5, 6) ). The substrate precursors include those of polypeptide hormones, neuropeptides, growth factors, growth factor receptors, certain plasma proteins, and viral envelope glycoproteins. Human furin, which is encoded by the fur gene, is ubiquitously expressed in all tissues and represents the first identified mammalian member of this family of convertases(7, 8) . The other mammalian convertases are the neural and endocrine-specific PC1 ( (9) and (10) ; also called PC3 in (11) ), PC2(9, 12) , the widely distributed PACE4(13) , PC5 ((14) , also called PC6 in (15) ), and the testis-specific PC4(16, 17) . Several of these mammalian enzymes were found to have counterparts in other species, such as the PC1-like (or PC3-like) protein from Hydra vulgaris (18) and anglerfish(19) , the PC1, PC2, and furin-like cDNAs of Aplysia californica(20, 21) , the PC2 and furin-like structures of Lymnaea stagnalis(22) , the furin-like bli-4 gene product of Caenorhabditis elegans(23) , Xen-14 and Xen-18 of Xenopus laevis(24) , and the furin-like enzymes of Drosophila melanogaster(25, 26, 27, 28) .

The process of alternative splicing with the consequent production of several mRNA transcripts has been demonstrated for several members of the pro-protein convertase family, including human (h) (^1)PACE4(13, 29) , rat (r) and mouse (m) PC4(16) , mPC5 (also called PC6)(14, 30) , Hydra PC1 (also called PC3)(18) , and Drosophila (d) dfurin1(28) . In D. melanogaster, the fur-like sequences dfurin1 and dfurin2 were reported to originate from two distinct genes(27, 28) . In addition, Northern blot analysis of Drosophila embryos revealed that expression of the Dfur1 gene generated four different sizes of transcripts encoding three proteins differing in their C-terminal sequence, which were called dfurin1, dfurin1-CRR, and dfurin1-X(28) . The predicted structural characteristics of the dfurin1-related enzymes are shown in Fig. 1, where they are compared to those of dfurin2, hfurin, mPC5 (also called PC6), and hPACE4. The dfurin1 isoforms are identical in structure from their N terminus up to their catalytic region but exhibit different structural domains in their C-terminal segments. Notably, the dfurin1-CRR isoform possesses a cysteine-rich domain that is also seen in dfurin2, hfurin, hPACE4, and mPC5 (also called PC6) (Fig. 1). Conservation of the Cys-rich motif in these various convertases suggests an important function for this segment of the molecule. However, complete deletion of this domain in mfurin did not seem to affect the capacity of this enzyme to intracellularly cleave coexpressed mutated (M2R) mouse pro-renin(31) . In an attempt to understand the functional importance of the C-terminal diversity of dfurin1-related enzymes, Roebroek et al.(28) coexpressed each of these convertases with either pro-von Willebrand factor or pro-beta(A)-activin as substrates. In that study, no significant differences with regard to the cleavage specificity of dfurin1, dfurin1-CRR, and dfurin1-X were observed. In contrast, although dfurin2 was able to efficiently process pro-von Willebrand factor, it was highly limited in its capacity to cleave pro-beta(A)-activin(28) . So far, aside from dfurin1 and dfurin2, no other convertases have been reported in Drosophila. In addition, the dfurin1 isoforms revealed non-overlapping tissue distribution during the various stages of embryonic development (28) and were found to be expressed within various organs, including the central nervous system and hindgut(26, 28) . This suggests widespread but distinct functions and/or cellular localization for each gene product and its isoforms. However, the endogenous Drosophila substrates are not yet known.


Figure 1: Alignment of Drosophila and mammalian pro-hormone convertases. Legend to protein domains is depicted at the bottom, and the amino acid length of each convertase is given at the right of the picture.



In this study, in order to define the cleavage selectivity of each convertase and its isoforms, we compared their catalytic properties to those of the mammalian enzymes PC1, PC2, and furin by cellular coexpression of vaccinia virus recombinants of dfurins with selected precursor substrates. This allowed us to probe whether the C-terminal variable segments of the dfurin1 isoforms can affect their cellular cleavage selectivity. Our selection of representative substrates was based on the classification proposed by Bresnahan et al.(32) , where precursors are subdivided into three categories: Type I precursors contain the consensus Arg-Xaa-(Lys/Arg)-Arg sequence at their cleavage site. These pro-proteins include growth factors and proteins usually processed within cells expressing furin and possibly PACE4 and/or PC5(7) . Type II precursors exhibit a pair of basic residues at the site of cleavage but no Arg residue at the P4 position. Representative examples include most pro-hormones, such as pro-opiomelanocortin (POMC), which is found in cells containing secretory granules and is now known to be processed in vivo by PC1 and PC2(33) . Finally, type III precursors are cleaved at a monobasic site usually represented by a single Arg such as the C-peptide cleavage site of pro-dynorphin(34) . Aside from defining the cleavage selectivity of each enzyme, we also characterized the biosynthetic products of each dfurin and defined their kinetics of synthesis and post-translational modifications.


MATERIALS AND METHODS

Construction of Recombinant Vaccinia Viruses

The dfurin1, dfurin1-CRR, and dfurin1-X cDNA inserts were excised from the plasmid pGEM11Zf(+) by initial XbaI digestion. The dfurin2 cDNA was excised from pGEM-3Zf(+) by digestion with RcaI. All of these linearized digestion products were blunted and subsequently digested with HindIII. These inserts were then ligated into a vaccinia virus transfer vector pMJ601 (35) linearized with SmaI/HindIII. The Dfur1-X cDNA insert was also excised from a pSVL vector and cloned into a pVV3-derived transfer vector(36) . Expression of dfurin1-X-PVV and the expression of dfurin1-X-PMJ are driven by a vaccinia late promoter (36) and a synthetic promoter(35) , respectively. The vectors containing the dfurin inserts were then used to generate recombinant vaccinia viruses (VV:dfur1, VV:dfur1-CRR, VV:dfur1-X, VV:dfur1-X.PVV, and VV:dfur2) as previously reported(33) .

Cellular Infections by Vaccinia Virus Recombinants

In this work we have used four types of cells, two of which do not have secretory granules and two of which contain dense core secretory granules: LoVo human colon carcinoma cells (American Type Tissue Collection), which do not express a functional furin (37) and do not contain secretory granules; BSC40 African green monkey kidney cells, which also do not contain secretory granules; and GH4C1 rat somatomammotroph cells and AtT-20 mouse anterior pituitary cells, both of which contain dense core secretory granules. Aside from the VV:dfurins, the other vaccinia viruses used in the present studies consisted of either the wild type virus (VV:wt) or the recombinants VV:mPC1, VV:mPC2(33) , VV:hfur(38, 39) , VV:mPOMC, VV:pro-m7B2(40) , and VV:rdynorphin(41) . All infections and coinfections were performed at a multiplicity of infection of 1 plaque forming unit (pfu)/cell for each virus used as described previously(33) .

Cellular Expression and Radiolabeling Studies

Biosynthetic analyses were performed as described previously(33, 38, 39) . Briefly, 17 h postinfection, cells were washed and then switched for 1 h to a methionine-free medium (RPMI 1640, Life Technologies, Inc.) supplemented with 0.5% fetal calf serum. Subsequently, cells were either pulselabeled with [S]methionine (100 µCi/ml) for 1 or 2 h or for 8 or 15 min and then chased for 45 or 60 min or for 120 min, respectively, in the presence of excess unlabeled methionine. In temperature block experiments, the cells were preincubated without methionine at 37 °C and then pulse-labeled with [S]methionine for 2 h at either 37 or 20 °C. In experiments using brefeldin A or tunicamycin, cells were preincubated and incubated with either brefeldin A or tunicamycin as described before(39) . In experiments with ionophore A23187, infected cells were preincubated in RPMI 1640 medium lacking methionine for 1 h and subsequently incubated in calcium-free medium containing 1 µl/ml of 5 mM A23187 dissolved in ethanol. At the end of the incubation period, the media were removed and cells were disrupted in lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 20 µg/ml phenylmethylsulfonyl fluoride) by incubation on ice for 20 min. The media and cell lysates were immunoprecipitated with various antisera, and the precipitates were analyzed by electrophoresis on SDS-polyacrylamide gels (SDS-PAGE) followed by autoradiography.

Immunoprecipitations and SDS-PAGE Analyses

All immunoprecipitations were performed as described before(33) . For dfurin1 and dfurin2(28) , 10 µl of the corresponding antibodies were used per ml of either medium or cell lysate. For POMC-derived peptides, the polyclonal antibodies used recognize either adenocorticotropin hormone (ACTH) (AT-1) or beta-endorphin (beta-END) (AT-2) containing peptides(33) . The N-terminal human furin polyclonal antibody was obtained by immunization of rabbits using an octopus-branched synthetic peptide approach already used for the PC1 N-terminal antibody(38, 39) . The peptide chosen consisted of the sequence Pro-Asp-Val-Tyr-Gln-Glu-Pro-Thr-Asp-Pro-Lys-Phe-Gln, representing residues 108-120 of rat furin(42) . The 7B2 polyclonal antibody used was directed against the conserved 23-39 sequence of human 7B2(43) . For each coexpression experiment, the levels of convertases synthesized as well as the secreted products of either POMC or pro-7B2 were evaluated in parallel by specific immunoprecipitations followed by analysis by SDS-PAGE. The immunoprecipitation products were resolved by SDS-PAGE on either 6% gels, for dfurins, or 15% gels, for mPOMC and m7B2 products, followed by autoradiography. For preparative purposes, immunoprecipitated dfurin1 proteins were resolved on 6% SDS-PAGE gels, which were sliced (1 mm). The eluted radiolabeled proteins were subjected to microsequence analysis on an Applied Biosystems model 470A sequenator as described(39, 40) .

Chromatography and Radioimmunoassays

Chromatography and radioimmunoassays were performed as described previously(34, 41) . Briefly, 17 h postinfection, LoVo cells coinfected with VV:rdynorphin, and each of the VV:dfurins were incubated in Ham's F-12 medium (Life Technologies, Inc.) for 3 h. The media (1.5 ml) were deposited on a Sephadex G-50 (Pharmacia) column (1.5 times 90 cm) equilibrated with 1% formic acid and 0.1% bovine serum albumin, as reported earlier(34) . The collected fractions (2 ml) were dried, resuspended in 1 ml of 1:1 methanol:HCl (v/v), and assayed for C-peptide immunoreactivity by a specific radioimmunoassay using a C-terminally directed antiserum(34) . Using trypsin-digested pro-dynorphin, which releases the C-peptide quantitatively, we estimated that this antiserum recognizes the free C-peptide about 35-fold better than when it is attached to pro-dynorphin.


RESULTS

Comparative Cleavage Specificity of dfurin Convertases

In order to compare the cleavage selectivity of the three dfurin1 isoforms with each other and with dfurin2, we selected three representative precursor substrates, pro-7B2 (type I), POMC (type II), and pro-dynorphin (type III), which were coexpressed with the dfurins using vaccinia virus as a cellular expression system. For pro-7B2 and pro-dynorphin, coinfection studies were performed in a constitutively secreting human colon carcinoma LoVo cell line(37) . This cell line, which is devoid of endogenous active furin, was chosen because we have recently shown that pro-7B2 is rapidly cleaved into 7B2 by furin (44) and that pro-dynorphin is synthesized and processed in adrenal cortex cells, which lack secretory granules(45) . For POMC, a precursor exclusively synthesized in regulated cells, we chose the rat somatomammotroph cell line GH4C1, in which we previously demonstrated the capacity of PC1, PC2, and furin to process POMC(39) . For each coexpression experiment, the amounts of convertases synthesized were evaluated by specific immunoprecipitations (see Fig. 5and Fig. 8).


Figure 5: Biosynthesis of Dfur1- and Dfur2-encoded proteins in LoVo and BSC40 cells. LoVo and BSC40 cells were infected with either VV:wt (wt), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were pulse-labeled with [S]methionine and immunoprecipitated with rabbit anti-dfurin1 (the first wt lane, dfur1, 1-CRR, and 1-X) or anti-dfurin2 (the second wt lane and dfur2) antisera as described under ``Materials and Methods.'' Both cells (A) and media (B) were immunoprecipitated. Molecular mass markers and the positions of the highest molecular forms detected of the dfurins are indicated.




Figure 8: Biosynthesis of Dfur1- and Dfur2-encoded proteins in GH4C1 and AtT-20 cells. GH4C1 and AtT-20 cells were infected with VV:wt (wt), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). 17 h postinfection, cells were preincubated in medium lacking methionine and then pulse-labeled with [S]methionine for 2 h and immunoprecipitated with rabbit anti-dfurin1 (the first wt lane, dfur1, 1-CRR, 1-X) or anti-dfurin2 (the second wt lane and dfur2) antisera as described under ``Materials and Methods.'' Both cells (A) and media (B) were immunoprecipitated. Molecular mass markers and the positions of the highest forms of dfurins detected are indicated.



Coexpression of pro-m7B2 and dfurins in LoVo Cells

The cleavage site of pro-m7B2 contains a pentabasic sequence Arg-Arg-Lys-Arg-Arg (40, 46) and fits the Arg-Xaa-(Lys/Arg)-Arg consensus type I precursor cleavage site(32) . As shown in Fig. 2, although PC2 is not capable of processing pro-m7B2 (30 kDa) into its mature form m7B2 (23 kDa), furin, and to a much lesser extent PC1, is able to do so as reported earlier(44) . In turn, each dfurin was as efficient as hfurin in completely converting pro-m7B2 into m7B2 (Fig. 2). These data demonstrate that the recombinant vaccinia virus of each dfurin expresses an active enzyme that resembles mammalian furin in its activity more than either PC1 or PC2. However, the overexpression of each dfurin with pro-m7B2 did not reveal a difference in their cleavage preference toward this typical type I precursor.


Figure 2: Analysis of proteolytic processing of pro-m7B2 by the dfurins. LoVo cells were coinfected with 1 pfu of VV:pro-m7B2 and 1 pfu of VV:wt (wt), VV:mPC1 (mpc1), VV:mPC2 (mPC2), VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were pulsed for 15 min with [S]methionine and chased for 45 min in the presence of excess unlabeled methionine. Media were then immunoprecipitated and resolved by electrophoresis as described under ``Materials and Methods.'' Molecular mass markers and the relative positions of pro- and mature m7B2 are indicated.



Coexpression of mPOMC and dfurins in GH4C1 Cells

To further evaluate potential differences in cleavage site specificity of the dfurins, we coexpressed each of these enzymes with mPOMC as a representative type II precursor substrate. In this type of precursor, the known in vivo cleavage sites contain simple pairs of basic residues such as Lys-Arg and Arg-Arg without an Arg. As previously demonstrated, mPOMC is cleaved into distinct peptide products by PC1 as compared with PC2(33) . ACTH and beta-lipotropin (beta-LPH) are produced by PC1 cleavage, whereas PC2 is needed to generate beta-END and alpha-melanotropin from the same precursor(33) , together with the trimming enzymes carboxypeptidase E (47) and peptidylglycine alpha-amidating monooxygenase(48) . In addition, overexpression of furin and mPOMC in GH4C1 cells generated products similar to those obtained with PC1(39) . Thus, mPOMC was a useful model for discriminating activities among the dfurins that are similar to either PC1/furin or PC2. GH4C1 cells were coinfected with recombinant vaccinia viruses expressing mPOMC and each of the dfurins (Fig. 3). Following infection, the cells were pulse-labeled with [S]methionine for 2 h. The media were then immunoprecipitated with anti-beta-END and anti-ACTH antibodies, and the products were separated by SDS-PAGE. The autoradiograms showed that, similar to hfurin and mPC1, the three dfurin1 isoforms and dfurin2 were capable of cleaving mPOMC into beta-LPH (Fig. 3A) and ACTH (Fig. 3B), albeit with varying efficiencies. As was established with pro-von Willebrand factor and pro-beta(A)-activin by Roebroek et al.(28) , dfurin1 seems to be the most efficient of the Drosophila convertases at cleaving mPOMC, followed by dfurin1-CRR and dfurin2, whereas dfurin1-X demonstrated a very weak POMC processing capability. The low activity of dfurin1-X can be explained in part by its lower expression levels under the infection conditions used, even with the PMJ construct, which expresses about 3-4-fold higher amounts of enzyme activity (Fig. 3, A and B) and protein (data not shown) versus the dfurin1-X-PVV construct. Our results show that dfurin2 is equivalent to dfurin1-CRR in its cleavage selectivity and ability to produce beta-LPH (Fig. 3A) and ACTH (Fig. 3B). Quantitation of the various products was obtained by scanning the relative band intensities and normalizing the values with respect to the known number of methionines in each product (e.g. 3 for mPOMC and 1 for both beta-LPH and beta-END). The results showed that beta-LPH represented 38, 22, 7, and 25% of the total immunoprecipitated POMC-related molecules produced by dfurin1, dfurin1-CRR, dfurin1-X-PMJ, and dfurin2, respectively. These values are lower than those obtained with mPC1 and hfurin, where beta-LPH represented 79 and 75% of the total immunoprecipitated proteins, respectively (Fig. 3A). Similarly, quantitation of the data in Fig. 3B revealed that glycosylated ACTH migrating as an 11-kDa peptide (33) represents 51, 32, 12, 4, 2, and 8% of the total ACTH immunoreactivity produced by mPC1, hfurin, dfurin1, dfurin1-CRR, dfurin1-X-PMJ, and dfurin2, respectively. This order of cleavage efficiency is similar to that deduced from the beta-LPH immunoprecipitations (Fig. 3A). In contrast to PC2, which produced 37% beta-END and an 11% alpha-melanotropin-like peptide, none of the dfurins produced either of these typical PC2-generated peptides (Fig. 3). Taken together, these data demonstrate that dfurins exhibit similarities to furin/PC1, but not to PC2, in their cleavage selectivity of POMC. As shown in Fig. 8, a semi-quantitative evaluation of the protein levels of the convertases by specific immunoprecipitation suggests that the degree of cleavage observed correlates with the levels of the convertases in which the pro-segment has been excised.


Figure 3: Analysis of proteolytic processing of mPOMC by the dfurins. GH4C1 cells were coinfected with 1 pfu of VV:mPOMC and 1 pfu of VV:mPC1 (mPC1), VV:mPC2 (mPC2), VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X-PVV (1-X-PVV), VV:dfurin1-X-PMJ (1-X-PMJ), or VV:dfurin2 (dfur2). Cells were pulsed for 2 h with [S]methionine. Media were then immunoprecipitated with either anti-beta-END antibody (AT-1) (A) or anti-ACTH antibody (AT-2) (B) and resolved by electrophoresis as described under ``Materials and Methods.'' Molecular mass markers and the relative positions of POMC, beta-LPH, and beta-END (A), and ACTH, glycosylated ACTH (ACTH*), joining peptide-ACTH intermediary processing product (JP-ACTH*), and alpha-melanotropin-like peptide (alpha-MSH) (B) are indicated. Wt/mPOMC negative control coinfection was also performed and immunoprecipitated with both anti-beta-END antibody (AT-1) and anti-ACTH antibody (AT-2). Only the anti-ACTH immunoprecipitation is shown here.



Coexpression of Rat Pro-dynorphin and dfurins in LoVo Cells

Finally, the potential discriminative cleavage specificities of the dfurins toward monobasic (type III) sites were investigated using rat pro-dynorphin as a substrate and a C-terminally directed antibody that recognizes preferentially the free C-peptide. The latter peptide, which represents the last 15 amino acids of pro-dynorphin, is generated by cleavage post of a single Arg residue from the C terminus of pro-dynorphin in the sequence Val-Val-Thr-ArgSer(34, 41) . Media of LoVo cells in which rat pro-dynorphin was coexpressed with each of the dfurins were separated on Sephadex G-50 and assayed for C-peptide immunoreactivity (Fig. 4A). Quantitation of the results (Fig. 4B) revealed that the relative efficiency of C-peptide cleavage by the various convertases as compared with mPC1 is 1, 1.8, 1.6, 1.9, 0.6, and 0.6 for mPC1, hfurin, dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2, respectively. These data showed that dfurin1 and dfurin1-CRR exhibited similar efficiencies as compared with hfurin toward cleavage at the monobasic site of pro-dynorphin, whereas dfurin1-X and dfurin2 were about 3-fold less efficient.


Figure 4: Analysis of proteolytic processing of rat pro-dynorphin by the dfurins. As shown in B, LoVo cells were coinfected with 1 pfu of VV:pro-rdynorphin and 1 pfu of VV:mPOMC (mPOMC), VV:mPC1 (mPC1), VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (dfur1-CRR), VV:dfurin1-X (dfur1-X), or VV: dfurin2 (dfur2). 17 h postinfection, cells were incubated for 3 h with unsupplemented Ham's F-12 medium. Media were collected and resolved by chromatography on Sephadex G-50, and fractions were assayed by radioimmunoassay for C-peptide immunoreactivity as described under ``Materials and Methods.'' An example of the G-50 elution profile assayed by radioimmunoassay is depicted in A, where fractions of the coinfection medium of VV:pro-rdynorphin and VV:dfurin1-CRR separated on G50 were assayed for C-peptide immunoreactivity. Arrows indicate the elution positions of rat pro-dynorphin (1) and C-peptide (2). In B, quantitation of the percent processing into C-peptide for each rat pro-dynorphin coinfection was determined by dividing the sum of the picograms/fraction immunoreactivity found in the C-peptide peak by the total immunoreactivity due to both pro-dynorphin and C-peptide. These percentages were then divided by the mPC1 percent of cleavage value, yielding relative cleavage efficiency values for the dfurin convertases with respect to mPC1.



Biosynthesis and Molecular Forms of the dfurins

Biosynthesis of dfurins in Constitutive LoVo and BSC40 Cells

Both LoVo and BSC40 cells were infected with VV: dfurin1, VV:dfurin1-CRR, VV:dfurin1-X, or VV:dfurin2, with VV:wt as control, and metabolically labeled with [S]methionine 17 h postinfection. The cell extracts and media were immunoprecipitated with either dfurin1- or dfurin2-specific antibodies(28) . The precipitates were then resolved by SDS-PAGE on a 6% polyacrylamide gel (Fig. 5). Except for dfurin1-X, all dfurins exhibited the presence of more than one immunoprecipitable protein both intracellularly (Fig. 5A) and in the medium (Fig. 5B). The estimated apparent molecular masses of the major bands vary slightly between both cell types and are 119 and 95 kDa for dfurin1, 153, 140, and 117 kDa for dfurin1-CRR, 163 kDa for dfurin1-X, and 200 and 178 kDa for dfurin2 in LoVo cells, while in BSC40 cells the molecular masses calculated were of 110 and 90 kDa for dfurin1, 147, 130, and 110 kDa for dfurin1-CRR, 155 kDa for dfurin1-X, and 191 and 162 kDa for dfurin2. The molecular masses given were calculated as averages of the values deduced following linear regression analysis of at least two immunoprecipitation SDS-PAGE analyses. It is important to note that the apparent molecular masses are similar in both the media and the cells and that all forms observed intracellularly are secreted. This suggests that, in LoVo and BSC40 cells, these proteins are not anchored to membranes and represent C-terminally processed products of the various dfurins.

In order to further define some of the protein forms observed in LoVo cells, we undertook pulse-chase experiments using short pulse periods to determine potential precursor-product relationships for dfurin1- and dfurin1-CRR-immunoprecipitated proteins (Fig. 6). As shown in Fig. 6A, after a pulse of either 1 or 8 min, we saw the formation of at least two forms of dfurin1, migrating with apparent molecular masses of 123 and 119 kDa. As shown in the upper panel of Fig. 6A, the immunoprecipitation of the dfurin-1 products is specific, because no bands were observed when we used a normal rabbit serum. Also, after only 1 min of pulse, we saw the formation of both the 123- and the 119-kDa forms, with the former disappearing within 30 min of chase. In the bottom panel, this 123 kDa form, which is absent after a 1-h chase, could represent the precursor of dfurin1 still containing a transmembrane domain (TMD), because it is never detected in the medium (data not shown). Similarly, the 119-kDa form would represent the precursor form lacking its TMD, because it is secreted into the medium (see Fig. 5B). Furthermore, the difference of 4 kDa observed between the 123- and 119-kDa forms cannot be due to an N-terminal truncation of the pro-segment, because we should have expected a variance of about 17 kDa between the calculated masses of pro-dfurin1 (88 kDa) and dfurin1 (71 kDa), assuming about 1.5 kDa/N-glycosylation site(25) . We note the slow migration of the putative TMD-containing pro-dfurin1, which travels with an apparent molecular mass overestimated by about 35 kDa (123 versus 88 kDa). Such abnormal migration on SDS-PAGE has previously been observed with a number of proteins, including human pro-furin and furin, which migrate with apparent molecular masses about 20 kDa higher than expected from their amino acid sequence(8, 49) . The microsequence of 2 times 10^6 cpm of the 119-kDa form of dfurin1 (in LoVo cells) labeled in [S]methionine did not reveal the presence of methionine residues within the first 20 cycles, in agreement with its tentative assignment as pro-dfurin1 (starting at residue 152). However, microsequencing of the protein equivalent to the 95-kDa form of dfurin1 (obtained from AtT20 cells; see Fig. 8) revealed methionines at positions 5, 15, and 17 in agreement with a protein sequence starting at residue 310 of dfurin1. This result demonstrates that the smallest dfurin1 form detected represents the mature enzyme obtained following the removal of its TMD and cleavage of the pro-segment at the Arg-Ser-Lys-Arg site.


Figure 6: A, pulse-chase analysis of dfurin1 and dfurin1-CRR. In the top panel, LoVo cells were infected with VV:dfurin1 (all lanes), and some cells were pulse-labeled with [S]methionine for 1 min (p1, p1 c10, and p1 c30) and then chased for 10 (p1 c10) or 30 min (p1 c30). Other cells were pulsed for 8 min (p8 adf1 and p8 aNRS). In p1, p1 c10, p1 c30, and p8 adf1, cells were immunoprecipitated with rabbit anti-dfurin1 antiserum, whereas in p8 aNRS, VV:dfurin1-infected cells were immunoprecipitated with normal rabbit serum. In the bottom panel, LoVo cells were infected with VV:wt (wt), VV:dfurin1 (dfurin1), or VV:dfurin1-CRR (dfurin1-CRR). Cells were pulse-labeled with [S]methionine for 8 min. Some cells were chased for 1 (p 8 min. c 1h) or 2 h (p 8 min. c 2h). All cells were then immunoprecipitated with rabbit anti-dfurin1 antiserum as described under ``Materials and Methods.'' Molecular mass markers are indicated. B, biosynthesis of dfurins in the presence of tunicamycin. BSC40 cells were infected with VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were pulse-labeled with [S]methionine in the presence of tunicamycin for 1 h and immunoprecipitated with rabbit anti-dfurin1 antiserum (dfur1, 1-CRR, and 1-X) or rabbit anti-dfurin2 antiserum (dfur2) as described under ``Materials and Methods.'' Molecular mass markers are indicated. C, biosynthesis of dfurins in the presence of ionophore A23187. BSC40 cells were infected with VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were pulse-labeled with [S]methionine in a calcium-free medium containing A23187 for 1 h and immunoprecipitated with rabbit anti-dfurin1 antiserum (dfur1, 1-CRR, and 1-X) or rabbit anti-dfurin2 antiserum (dfur2) as described under ``Materials and Methods.'' Molecular mass markers are indicated.



When dfurin1-CRR was pulse-labeled for 8 min followed by a chase of 1 and 2 h (Fig. 6A), we observed the formation of a major 153-kDa form and the transient production of a minor 162-kDa protein that disappears after a chase of 1 h. Using similar arguments to those used for dfurin1, the 162-kDa form likely represents the pro-dfurin1-CRR with its TMD, whereas pro-dfurin1-CRR lacking the TMD may be represented by the 153-kDa form. Here also, the 11-kDa difference in masses between these forms (average of four separate experiments) is smaller than that expected from the loss of the N-terminal pro-segment, for which a shift of about 17 kDa would be expected (113 and 96 kDa for pro-dfurin1-CRR and dfurin1-CRR, respectively)(28) .

As shown in Fig. 6B, treatment of VV:dfurin-infected BSC40 cells with tunicamycin reveals that all dfurins are N-glycosylated. The apparent molecular masses of the major forms are 105, 139, 145, and 178 kDa for dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2, respectively. Accordingly, in the presence (Fig. 6B) and the absence (Fig. 5A) of tunicamycin, the observed molecular masses of the major intracellular forms differ by about 5, 8, 10, and 13 kDa for dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2, respectively. It is interesting to note that in the presence of tunicamycin, we also detected small amounts of larger molecular forms migrating at 118, 150, and 157 kDa for dfurin1, dfurin1-CRR, and dfurin1-X, respectively. These may represent the pro-forms still containing the TMD. In addition, we note that prevention of N-glycosylation causes the appearance of excessive degradation products, as was originally reported for PC1 and PC2, for which such degradation was shown to occur within the endoplasmic reticulum (ER)(39) .

Finally as shown in Fig. 6C, in BSC40 cells infected with VV:dfurins in the presence of the Ca ionophore A23187, only the putative pro-dfurins lacking the TMD are detectable by immunoprecipitation, because the molecular masses of the major forms are virtually the same as those seen in a similar 1-h pulse in the absence of this Ca-depleting agent (compare Fig. 5A and Fig. 6C). Because we could not detect the forms that presumably lack their pro-segment in the presence of A23187, this suggests that the cleavage of the pro-sequence is largely inhibited under conditions of low Ca concentrations. In contrast, because we did not observe the higher molecular mass forms still containing the TMD, this implies that A23187 does not significantly affect the removal of this C-terminal domain.

In order to define whether the shorter forms of the dfurins are produced within the ER/Golgi stacks, we repeated the pulse labeling of LoVo cells infected with the various vaccinia virus recombinants of dfurins both at the permissive 37 °C and restrictive 20 °C temperature, as well as in the presence of the fungal metabolite brefeldin A. It is well known that at 20 °C, the transport of membrane glycoproteins from the trans-Golgi network (TGN) to the cell surface is severely retarded(50) , whereas brefeldin A causes the redistribution of the cis- and medial Golgi stacks to the ER(51) , thus preventing traffic from this compartment to the TGN. The data in Fig. 7A demonstrates that after a 2-h pulse with [S]methionine performed at 37 or 20 °C, the processing pattern of the dfurin1 isoforms and dfurin2 is very similar at both temperatures. No immunoreactive dfurin1- or dfurin2-related proteins are detected in the medium at 20 °C, confirming that secretion is blocked at this temperature (data not shown). This suggests that all observed processing events occur while the proteins transit from the ER up to the TGN and not during or after secretion. However, in the presence of brefeldin A (Fig. 7B), the protein forms detected are mostly the high molecular mass pro-forms lacking the TMD with small amounts of shorter forms of dfurin1-CRR and dfurin2 detectable, suggesting that the removal of the pro-segment of the dfurins occurs in the TGN but can also happen earlier for dfurin1-CRR and dfurin2.


Figure 7: A, comparative biosynthesis of Dfur1- and Dfur2-encoded proteins at 37 and 20 °C. LoVo cells were infected with VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), or VV:dfurin2 (dfur2). Cells were pulse-labeled with [S]methionine for 2 h at either 37 or 20 °C as indicated and immunoprecipitated with anti-hfurin (hfur), anti-dfurin1 (dfur1 and 1-CRR), or anti-dfurin2 (dfur2) antisera as described under ``Materials and Methods.'' Molecular mass markers are indicated. B, biosynthesis of dfurin1, dfurin1-CRR, and dfurin2 in the presence of brefeldin A. LoVo cells were infected with VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), or VV:dfurin2 (dfur2). Cells were pulse-labeled with [S]methionine in the presence of brefeldin A for 1 h and immunoprecipitated with anti-dfurin1 (dfur1 and 1-CRR) or anti-dfurin2 (dfur2) antisera as described under ``Materials and Methods.'' Both cells and media were immunoprecipitated. Molecular mass markers are indicated.



Biosynthesis of dfurins in GH4C1 and AtT-20 Cells

In order to also define the molecular forms of dfurins obtained in regulated cells and compare them with those previously observed in constitutive cells, GH4C1 and AtT-20 cells infected with either VV:dfurins or VV:wt were pulse-labeled for 2 h with [S]methionine. Fig. 8depicts the autoradiogram of the SDS-PAGE separation of the immunoprecipitated products obtained from the cell extracts (Fig. 8A) and media (Fig. 8B). The results show that in GH4C1 and AtT-20 cell extracts, the processed products of each dfurin are similar in size to those detected in BSC40 and LoVo cells (compare Fig. 8A and Fig. 5A). In AtT-20 and GH4C1 cells, we detect several molecular forms of respective masses: (105, 115) and (90, 94) kDa for dfurin1, (147, 150), (130, 133), and (110, 113) kDa for dfurin1-CRR, 155 kDa for dfurin1-X, and (191, 195) and 160 kDa for dfurin2. In the media of GH4C1 cells, only the 94-, 113-, and 160-kDa forms of dfurin1, dfurin1-CRR, and dfurin2 are observed, respectively, whereas no dfurin-X products were detected. Similarly, in AtT-20 media, the proteins detected are: the 90-kDa form of dfurin1, the 110-kDa form of dfurin1-CRR (with smaller amounts of the 147- and 130-kDa forms), no dfurin1-X, and mostly the 160-kDa form of dfurin2 with very small amounts of the 191-kDa form. Therefore, in contrast to the results obtained with constitutive cells (Fig. 5B), in the media of GH4C1 and AtT-20 cells we observed only the smaller forms of the dfurins (Fig. 8B).


DISCUSSION

Although it has been established that multiple mRNA forms exist for each one of the six known mammalian convertases belonging to the kexin/subtilisin family(3, 4, 5, 6, 52, 53) , so far only the predicted protein sequences of PACE4(13, 29) , PC4(16, 54) , and PC5 (also called PC6) (30) have been reported to be affected by this diversity. The physiological advantage provided by this C-terminal diversity is not yet known, especially because the complete deletion of the C-terminal Cys-rich segments of mfurin (31) does not seem to affect the intracellular cleavage capability of this enzyme in the TGN. Conceivably, the different C-terminal sequences present in the various isoforms may impart a specific cellular address, as is the case for the amidation enzyme isoforms(55) . Alternatively, the rate of processing of potential substrates or the cleavage selectivity of the various isoforms for different precursors may be affected by their C-terminal diversity. The availability of three different dfurin1 isoforms (28) allowed us to address this question in the context of mammalian cells. The identification of a second furin-like enzyme in Drosophila, dfurin2(27) , gave us the opportunity to compare the cleavage selectivity of dfurin1 and dfurin2 convertases with three types of precursors, each exhibiting a different processing motif. Our results using pro-7B2 and those of Roebroek et al.(28) using pro-von Willebrand factor, both of which contain the consensus cleavage motif Arg-Xaa-(Lys/Arg)-Arg, show that all dfurins are able to process type I precursors. Our data extend the characterization of the dfurins to show that these convertases exhibit cleavage selectivities closer to those of mammalian furin or PC1, but not PC2, toward either type II (POMC) or type III (pro-dynorphin) precursors. Although quantitative differences in the amounts of generated products were noted between each dfurin1 isoform, we cannot exclude the fact that some of these variations are not due to the expression of different levels of these enzymes, especially in the case of dfurin1-X. Indeed, as shown in Fig. 3and Fig. 8as well as in the microsequencing results, it appears that the degree of processing of POMC can be directly correlated with the levels of immunoprecipitated convertases in which the pro-segment has been excised. In addition, coexpression studies with pro-7B2 (Fig. 2) and pro-dynorphin (Fig. 4) show that dfurin1 and dfurin1-CRR are closer to mammalian furin rather than to PC1 in their substrate cleavage efficacy. Our data with pro-7B2 (Fig. 2) and POMC (Fig. 3) demonstrated that dfurin2 exhibits similar cleavage efficiency and selectivity to dfurin1 and dfurin1-CRR. In contrast, the extent of cleavage of pro-dynorphin by dfurin2 is similar to that of dfurin1-X and about 40% of that observed for dfurin1 and dfurin1-CRR (Fig. 4). This difference in cleavage efficiency of dfurin1-X and dfurin2 toward rat pro-dynorphin compared with pro-m7B2 is substrate-determined and not cell typedetermined, because both studies were conducted in LoVo cells. A similar lower efficiency of cleavage of pro-beta(A)-activin by dfurin1-X as compared with the two other dfurin1 isoforms was reported earlier(28) , whereas dfurin2 did not cleave this substrate but processed pro-von Willebrand factor(28) . (^2)Thus, the C-terminal domain of the dfurin1 isoforms does not affect their cleavage selectivity. In no case did these convertases exhibit a PC2-like processing pattern. It is therefore unlikely that the protein diversity generated by the differential splicing of the Dfur1 gene can generate an enzyme with a similar cleavage preference as that of PC2, suggesting that the true Drosophila PC2-like convertase has yet to be identified. In this regard, a PC2-like convertase was recently cloned from C. elegans, an organism more evolutionarily ancient than D. melanogaster(56) .

Because the C-terminal variability of the dfurin1 proteins does not affect their cleavage specificity, is it possible that this domain influences the biosynthetic transformations undergone by the dfurins? To answer this question, we examined the fate of these proteins in several cell types. Originally, Roebroek et al.(28) reported two forms of dfurin1 of approximate molecular masses of 115 and 110 kDa in COS cells transfected with a dfurin1 cDNA. In the two constitutive cells used in this vaccinia virus infection study, LoVo and BSC40, using the same antibodies as Roebroek et al.(28) , we were able to detect up to three forms of dfurin1 with molecular masses of 123 (Fig. 6A), 119 and 95 kDa (Fig. 5B), with the longest form only observed when short pulse periods were used (Fig. 6A). It is likely that the two shorter forms are equivalent to those reported in the COS cells study (28) . Because our data show that these smaller two forms are secreted into the medium and that microsequencing confirmed their assignment as pro-dfurin1 and dfurin1, the 123-kDa form should represent pro-dfurin1 with its TMD, a form that rapidly disappears during the chase (Fig. 6A). The proposed cleavage of the 123-kDa form of dfurin1 generating the soluble smaller proteins and occurring N-terminal to the TMD seems to be unaffected by the presence of either the Ca ionophore A23187 (Fig. 6C) or brefeldin A (Fig. 7B). This suggests that the C-terminal TMD cleavage occurs early along the biosynthetic pathway, most probably within the ER. Because treatment with A23187 inhibits the formation of dfurin1, the Ca dependence of the TMD cleavage reaction is not the same as that of the pro-dfurin1 to dfurin1 processing. The same conclusion was drawn for the other dfurins.

Similar to the results with dfurin1, our results with dfurin1-CRR demonstrate the production of a transient cellular 162-kDa form (Fig. 6A), which we believe represents pro-dfurin1-CRR with its TMD, whereas the other soluble forms represent pro-dfurin1-CRR, dfurin1-CRR, and a shorter C-terminally truncated form, respectively, all lacking the TMD because they can be detected extracellularly. Both the dfurin1-X and dfurin2 proteins that immunoprecipitated were likewise found in the media of infected cells. This implies that the TMD of all dfurins is rapidly removed, yielding a pro-dfurin form that is then further processed to dfurin. Only in the case of dfurin1-CRR did we observe a third smaller form in both cells and media. Although the processing sites that result in TMD cleavage of the dfurins are difficult to define in view of their abnormal migration on SDS-PAGE, we observed that this shortening of the dfurins is not Ca-dependent and can occur in LoVo cells, which are devoid of endogenous hfurin activity(37) . When hfurin and mPC6-B (30) are overexpressed in cell lines, soluble forms are also observed arising from the partial loss of their TMD. However, this shedding event occurs after the exit of hfurin and mPC6-B from the ER, possibly within the TGN or at the cell surface(49, 57) . (^3)

The brefeldin A and temperature block experiments demonstrate that the processing of pro-dfurins to dfurins occurs in the TGN, whereas the zymogen cleavage of mammalian pro-furin to furin has been demonstrated to occur in the ER(49) . Only in the case of dfurin1-CRR and dfurin2 did we observe some processing in the presence of brefeldin A (Fig. 7B). Could it be that the furin-like convertases endowed with Cys-rich motifs can undergo an earlier pro-segment removal, as is the case with dfurin1-CRR, dfurin2, and mammalian furin, whereas those lacking this structural characteristic, such as dfurin1, dfurin1-X, and PACE4C, undergo later processing? Future work on the definition of the functional role of the Cys-rich motif in convertases will undoubtedly shed more light on this question.

In regulated cells, such as AtT-20 and GH4C1, we observed an intracellular pattern of immunoprecipitated proteins (Fig. 8A) similar to that seen in the constitutive cells. In contrast, in the media of AtT-20 and GH4C1 cells, the smaller forms of dfurins are predominantly secreted (Fig. 8B), especially for dfurin1-CRR and dfurin2(26, 27, 28) . Thus, the presence of the Cys-rich motif may not only confer to dfurin-CRR and dfurin2 the ability to undergo earlier zymogen processing along the secretory pathway but also an increased probability of C-terminal cleavage and secretion in regulated cells.

In conclusion, the data presented in this work showed that the three isoforms of dfurin1 and dfurin2 have similar characteristics to mammalian furin in terms of both catalytic activity and loss of their TMD. The differences in the C-terminal structure of the dfurin1 isoforms do not seem to influence their catalytic activity but may affect the rate and cellular site of processing of their pro-segment and ultimately influence their residence in different organelles. It is therefore possible that each isoform that is expressed in a tissue-specific manner at different stages of the Drosophila embryonic development (26, 28) may exert actions on specific substrates, which are coordinately expressed. Some of the possible Drosophila precursor substrates that are cleaved at paired basic residues include those related to the transforming growth factor-beta family, such as the decapentaplegic protein(58) , the integrin alpha-chain(59) , and insulin-like pro-receptor(60) . The conclusions drawn from the results of this work may well be applicable to the mammalian PC5 (also called PC6)(14, 30) , PACE4(29) , and PC4 (16) , because these proteins also exhibit several isoforms, some of which contain the same Cys-rich motif as that found in dfurin1-CRR (26, 28) and dfurin2(27) . The results obtained from this study should help to broaden our understanding of the role of differentially spliced forms of pro-protein convertases both in mammalian and non-mammalian systems, where such diversity has also been reported (for reviews see Refs. 4, 5, and 52). Future work will undoubtedly lead to a more detailed definition of the roles of the C-terminal domains of these varied pro-protein and pro-hormone convertases.


FOOTNOTES

*
This work was supported by the Medical Research Council of Canada Grant PG11474 (to N. G. S. and M. C.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: J. A. DeSève Laboratory of Biochemical Neuroendocrinology, Clinical Research Inst. of Montreal, 110 Pine Ave. West, Montreal, Quebec H2W 1R7, Canada. Tel.: 514-987-5609; Fax: 514-987-5542.

(^1)
The abbreviations used are: h, human; POMC, pro-opiomelanocortin; r, rat; m, mouse; d, Drosophila; pfu, plaque-forming unit; PAGE, polyacrylamide gel electrophoresis; TMD, transmembrane domain; ER, endoplasmic reticulum; TGN, trans-Golgi network; beta-LPH, beta-lipotropin hormone; beta-END, beta-endorphin.

(^2)
Roebroek, A. J. M., Ayoubi, T. A. Y., Creemers, J. W. M., Pauli, I. G. L., and Van de Ven, W. V. M.(1995) DNA Cell Biol., in press.

(^3)
S. Benjannet and N. G. Seidah, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Gary Thomas (Vollum Institute, Portland, OR) for his generous gift of the VV:mPOMC recombinant vaccinia virus construct and Dr. Ajoy Basak (Clinical Research Institute of Montréal, Canada) for synthesizing the peptide used to procure the hfurin antibody. Many thanks to Suzanne Benjannet for sharing her PC5 data and her expertise of the vaccinia expression system, to Drs. Florence Vollenweider and Didier Vieau for helpful comments and friendly discussions, as well as to Normand Rondeau for preparation of some of the dfurin vaccinia viruses. The technical assistance of Aida Mammarbachi, Odette Theberge, and Danielle Sorel is appreciated. The secretarial assistance of Lucie Houle is acknowledged.


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