©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Processing of Proglucagon by the Subtilisin-like Prohormone Convertases PC2 and PC3 to Generate either Glucagon or Glucagon-like Peptide (*)

(Received for publication, July 13, 1995; and in revised form, August 17, 1995)

Yves Rouillé(§) Sean Martin Donald F. Steiner (¶)

From the Department of Biochemistry and Molecular Biology and The Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proglucagon is processed differently in the islet alpha cells and the intestinal endocrine L cells to release either glucagon or glucagon-like peptide 1-(7-37) (GLP1-(7-37)), peptide hormones with opposing actions in vivo. In previous studies with a transformed alpha cell line (alphaTC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical alpha cell pattern of proglucagon processing, giving rise to glucagon and leaving unprocessed the entire C-terminal half-molecule known as major proglucagon fragment or MPGF (Rouillé, Y., Westermark, G., Martin, S. K., Steiner. D. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3242-3246). Here we present evidence, using mouse pituitary AtT-20 cells infected with a vaccinia viral vector encoding proglucagon, that PC3 (SPC3), the major neuroendocrine prohormone convertase in these cells, reproduces the intestinal L cell processing phenotype, in which MPGF is processed to release two glucagon-related peptides, GLP1 and GLP2, while the glucagon-containing N-terminal half-molecule (glicentin) is only partially processed to oxyntomodulin and small amounts of glucagon. Moreover, in AtT-20 cells stably transfected with PC2 (AtT-20/PC2 cells), glicentin was efficiently processed to glucagon, providing further support for the conclusion that PC2 is the enzyme responsible for the alpha cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and betaTC-3), proglucagon was also processed extensively to both glucagon and GLP1-(7-37), although STC-1 cells express lower levels of PC2 and processed the N-terminal domain to glucagon less efficiently. In contrast, GH(4)C(1) and COS 7 cells, which express very little or no PC2 or PC3, failed to process proglucagon, aside from a low level of interdomain cleavage which occurred only in the GH(4)C(1) cells. In vitro PC3 did not cleave at the single Arg residue in GLP1 to generate GLP1-(7-37), its truncated biologically active form, indicating the likelihood that another convertase is required for this cleavage.


INTRODUCTION

The discovery of a novel family of mammalian proteases related to the yeast processing enzyme kexin and the bacterial serine protease subtilisin has provided new insights into the cellular mechanisms of precursor processing in the secretory pathway(1, 2, 3) . Recent studies have suggested that two members of this six-member family, PC2 (SPC2) and PC3 (SPC3; also called PC1), are the key enzymes involved in the proteolytic processing of a large variety of neuroendocrine precursors in the brain and many endocrine tissues throughout the body in which these enzymes are predominantly localized(1, 3) . PC2 and PC3 both participate in the processing of proinsulin to insulin in the pancreatic beta cell (4) and of POMC (^1)to ACTH and/or other products in the pituitary(5, 6) . Moreover, it has been demonstrated that the known differential processing of POMC to different products in the anterior versus intermediate lobes of the pituitary (7) is due to the differential expression of PC2 and PC3 in these lobes (8) . Attention is thus focused on defining the potential role of these and/or other cellular proteases in the differential processing of other important neuroendocrine precursors such as proglucagon.

The 18-kDa mammalian proglucagon protein contains three homologous hormonal sequences, glucagon, glucagon-like peptide 1 (GLP1), and glucagon-like peptide 2 (GLP2), separated by two intervening peptides, IP-1 and IP-2, and preceded by an N-terminal extension called glicentin-related polypeptide (GRPP) (Fig. 1). These peptides are all linked by pairs of basic amino acids (Lys-Arg or Arg-Arg), that are used as cleavage sites during the processing. In mammals, the same precursor is initially synthesized in the alpha cells of the islets of Langerhans in the pancreas and in the endocrine L cells of the intestinal mucosa(9, 10) ; however, differential processing results in the formation of different sets of peptides with opposing biological activities. The pancreatic alpha cell secretes glucagon, which stimulates glycogenolysis and gluconeogenesis in the liver and counterbalances the hypoglycemic action of insulin, whereas the intestinal L cell secretes a very potent insulinotropic hormone, recently identified as GLP1-(7-37), a truncated form of GLP-1(11, 12) .


Figure 1: Proglucagon differential processing in the pancreas and the intestine. The primary structure of proglucagon is represented with glucagon, glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2) sequences in hatched boxes. The upper part shows the peptides resulting from proglucagon processing in the pancreatic alpha cell. These peptides are the glicentin-related polypeptide (GRPP, proglucagon-(1-30)), glucagon (proglucagon-(33-61)), the intervening peptide 1 (IP-1, proglucagon-(64-69)), and the major proglucagon fragment (MPGF, proglucagon-(72-158)). MPGF is partially processed to GLP-1 (proglucagon-(72-107)). The lower part shows the peptides resulting from proglucagon processing in the intestinal L cell. These peptides are glicentin (proglucagon-(1-69)), truncated GLP-1 (tGLP-1, proglucagon-(78-107)), intervening peptide 2 (IP-2, proglucagon-(111-122)), and GLP-2 (proglucagon-(126-158)). Glicentin is partially processed to GRPP and oxyntomodulin (PG 33-69). The cleaved sites are indicated by arrows, with the sequence surrounding the site shown above or below the arrow. Partially processed sites are indicated by dashed arrows. Note that the monobasic cleavage at Arg occurs only in the intestinal L cell.



The pancreatic alpha cell processes proglucagon mainly to glucagon, GRPP, IP-1, and MPGF (proglucagon-(72-158)), a 10-kDa peptide encompassing the GLP-1, IP-2, and GLP-2 sequences(13, 14) . A small fraction (10 to 20%) of the MPGF is further processed to GLP-1(15) . Low amounts of glicentin (proglucagon-(1-69)) have also been detected in the pancreas (16) , suggesting that it is an intermediate in the formation of glucagon. This processing pathway has been demonstrated recently through pulse-chase experiments in alphaTC1-6 cells, an islet-derived cell line transformed with SV40 large T antigen(17) . Proglucagon is initially cleaved at the Lys-Arg site to produce glicentin and MPGF. Glicentin is later processed at the Lys-Arg and Lys-Arg sites to yield GRPP, glucagon, and IP-1, while MPGF accumulates and is only slowly and partially processed to GLP-1-(1-36)-amide (proglucagon-(72-107)), after cleavage at the Arg-Arg site. Thus, the pancreatic processing of proglucagon occurs with an initial interdomain cleavage, followed by efficient processing of the N-terminal domain (glicentin) and very little processing of the C-terminal domain (MPGF).

In contrast, in the intestinal L cells, proglucagon processing results in the efficient formation of GLP-1, IP-2, and GLP-2(18) . Glucagon levels are very low in the intestine(19) . Rather, the N-terminal domain remains incompletely processed in the form of glicentin (20) and is only partially cleaved into GRPP and oxyntomodulin (proglucagon-(33-69))(21) , a form of glucagon C-terminally extended with IP-1(22) . An additional cleavage occurs at a single arginine residue within the GLP-1 sequence (Arg), yielding shortened active forms of GLP-1, GLP-1-(7-37) (proglucagon-(78-108)) and its desglycyl, C-terminally amidated counterpart, GLP-1-(7-36)-amide (proglucagon-(78-107)), collectively known as truncated GLP-1, or tGLP-1(12) . The processing of proglucagon in intestinal L cells thus may involve an initial interdomain cleavage, probably at the same site as observed in the pancreas, and is followed by extensive processing of only the C-terminal domain.

The simplest explanation for the alternative processing of the N- or C-terminal domains of glucagon, after their cleavage at the interdomain processing site, would be that it is due to the expression of different convertases in the alpha and L cells. This view is supported by recent findings that pancreatic alpha cells express high levels of PC2 and low levels, if any, of PC3(17, 23, 24) , whereas intestinal L cells contain immunoreactive PC3 but not PC2(25) . This suggests that PC2 generates the pancreatic phenotype while PC3 may be largely responsible for the intestinal phenotype. This hypothesis is also supported by the results of the present study correlating proglucagon processing in cell lines with differing levels of endogenous PC2 and PC3 convertases. We have examined the processing of proglucagon in a number of endocrine and non-endocrine-derived cell lines and have correlated the observed processing patterns with the levels of expression of the prohormone convertases PC2 and PC3. The results support the conclusions that PC3 is responsible for the processing of the C-terminal domain of proglucagon to release GLP1, and they indicate that another, as yet unidentified protease may be required for the conversion of GLP1 to tGLP1.


MATERIALS AND METHODS

Antisera

Glu 001 monoclonal antibody was obtained from Novo-Nordisk. Other antisera against glucagon were kindly provided by Dr. K. Polonsky (University of Chicago) and against GLP-1 and GLP-2 by Dr. C. Ørskov and Dr. J. J. Holst (Panum Institute, Copenhagen). These antisera have been used for immunoprecipitations in a previous study (17) . N-terminal specific PC3 antiserum 2B6 was a generous gift from Dr. I. Lindberg (Louisiana State University, New Orleans). PC2 antiserum PC2pep4 was raised against synthetic degenerate peptides corresponding to the sequence 611-635 of both human and mouse PC2.

Cell Culture

AtT-20 cells and COS 7 cells were grown in DMEM supplemented with 10% FBS. AtT-20/PC2 cells (26) were kindly provided by Dr. R. Mains (Johns Hopkins, Baltimore) and grown in DMEM supplemented with 20% FBS and 0.5 mg/ml G418 (Life Technologies, Inc.). betaTC3 cells were cultured in DMEM supplemented with 10% FBS and 10 mM HEPES buffer. STC-1 cells were generously donated by Dr. D. Hanahan (University of California, San Francisco) and grown in DMEM supplemented with 12.5% horse serum, 2.5% FBS, and 10 mM HEPES buffer. GH(4)C(1) cells were cultured in Ham's F-10 medium supplemented with 10% FBS. All media contained 2 mM glutamine, 50 units/ml penicillin G, and 50 µg/ml streptomycin sulfate. Cells were cultured at 37 °C in a 5% CO(2), 95% air mixture in a humidified incubator.

Vaccinia Virus

In order to construct a recombinant vaccinia virus expressing proglucagon, a hamster preproglucagon cDNA (generously donated by Dr. G. Bell, University of Chicago) was cloned into the plasmid pVZneo (kindly provided by Dr. G. Thomas, Vollum Institute, Portland). A 980-base pair fragment encompassing the entire coding sequence of the preproglucagon cDNA was recovered from the plasmid pshglu1 by DdeI restriction, purified by agarose gel electrophoresis, blunt-ended with Klenow, and cloned into BamHI-linearized, blunt-ended pVZneo. In the resulting pVZglu2 plasmid, the preproglucagon cDNA is positioned downstream from a P7.5 viral promoter. The orientation was verified by sequencing. The plasmid was used to construct a recombinant vaccinia virus by standard methods(27) . The recombinant virus VV:GLU expressed a 1.7-kilobase preproglucagon mRNA of the expected size, after infection in COS 7 cells (not shown), and a 19-kDa proglucagon recognized by anti-glucagon, anti-GLP-1 (see ``Results''), and anti-GLP-2 (not shown) antisera. For proglucagon expression, cells were grown to near confluence (except for GH(4)C(1) cells) in 6-cm plates. Infections were carried out at a multiplicity of infection = 1 (10 for betaTC3 cells) in 0.5 ml of DMEM at 37 °C for about 30 min (90 min for betaTC3 cells). Cells were returned to their normal growth medium for 6 h before metabolic labeling.

Metabolic Labeling

Cells were labeled at 37 °C for 12 h as described previously in 1 ml of serum-free DMEM lacking either methionine (for glucagon immunoprecipitation), or leucine and phenylalanine (for GLP-1 immunoprecipitation), and containing the corresponding amino acid(s) labeled with S or ^3H (0.5 mCi per dish; [S]Met = 1000 Ci/mmol and [^3H]Leu or -Phe = 130 Ci/mmol; from Amersham) and supplemented with 0.25% BSA and Trasylol at 500 units/ml (17) . Media were collected, centrifuged at 15,000 times g for 10 min, and kept at -80 °C if not used immediately. Cells were washed twice with cold D-PBS (D-PBS is 138 mM NaCl, 2.7 mM KCl, 1.2 mM KH(2)PO(4), 8.1 mM Na(2)HPO(4)), scraped off the plate, pelleted, and resuspended in 0.5 ml of immunoprecipitation buffer (50 mM Na(2)HPO(4), 2.5 µg/ml poly(L-lysine), 1 mg/ml BSA, 1 mM EDTA, 0.1% Triton X-100, 0.5% Nonidet P-40, and 0.9% NaCl, pH 7.4). Protease inhibitors were added to the cell suspensions and media to reach 0.1 mM 1,10-phenanthroline, 0.1 mM 3,4-dichloroisocoumarin, 20 µM E64, and 10 µM pepstatin. The cells were disrupted by sonication and centrifuged for 10 min at 15,000 times g. The supernatants were used for immunoprecipitation.

For some experiments with betaTC3 and COS 7 cells, the cell pellet was resuspended in 1 M acetic acid and extracted for 10 min at 95 °C. The extract was concentrated in a SepPak C18 cartridge (Millipore), and the peptides were eluted with 60% acetonitrile, 0.1% trifluoroacetic acid. The peptide extract was freeze-dried, redissolved in 20 µl of water, adjusted to 200 µl with immunoprecipitation buffer, incubated for 30 min on ice, and centrifuged for 15 min at 15,000 times g to remove insoluble materials. The whole supernatant was used for immunoprecipitation. Control experiments with alphaTC1-6 cells showed a similar recovery of the glucagon-containing peptides with both extraction methods (not shown). Recovery of I-labeled glucagon by these methods was greater than 80%.

Immunoprecipitations

Immunoprecipitations were performed on 200 µl of media or of cell lysates or extracts. The solution was precleared with 2 µl of normal rabbit serum and 50 µl of a 10% suspension in immunoprecipitation buffer of IgSorb (The Enzyme Center, Malden, MA), previously washed and decanted. After a 1-h incubation at 4 °C, the suspension was centrifuged, and the supernatant was transferred to a Microfuge tube containing 2 µl of the selected antiserum or 5 µg of monoclonal antibody and incubated for 16 h at 4 °C. Then, 30 µl of protein A-Sepharose (Pierce) was added, and the mixture was incubated for at least 1 h more at 4 °C on a rocking plate. The immune complexes bound to the beads were washed at least three times with 500 µl of immunoprecipitation buffer, once with 1% Triton X-100 in PBS, and once in TAS buffer (50 mM Tris-Cl, 100 mM NaCl, 0.25% BSA, pH 7.6) The final pellets were incubated for 5 min at 95 °C in 50 µl of sample buffer (50 mM Tris-Cl, 4% SDS, 12% glycerol, 0.01% bromphenol blue, 3% beta-mercaptoethanol, pH 6.8), and the supernatant was analyzed by SDS-PAGE (28) and fluorography.

Radiosequencing

GLP-1 was immunoprecipitated with carboxyamidated specific antiserum 89-390 from a lysate of AtT-20 cells infected with VV:GLU and radiolabeled with [^3H]Phe. Peptides were eluted from the beads with two 100-µl portions of 0.1 M HCl and injected into a Vydac C(18) reversed-phase HPLC column, along with 2 µg each of synthetic GLP-1-(7-36)-amide and GLP-1-(7-36)-amide, as carriers. The peptides were eluted at 1 ml/min with a linear gradient of acetonitrile (0-60% over 60 min) in trifluoroacetic acid (0.05%). Aliquots (10 µl) of each fraction (500 µl) were assayed for radioactivity in a liquid scintillation counter. A single peak of radioactivity was detected (fraction 89) that co-eluted with the synthetic peptides. The material contained in fraction 89 was submitted to 30 cycles of Edman degradation in a Beckman protein sequenator. Anilinothiazolinone derivatives were collected at each cycle, dried, redissolved in 0.5 ml of n-butyl chloride, and mixed with 10 ml of Optifluor-O (Packard), and the radioactivity was assayed in a liquid scintillation counter.

Immunoblot Analysis

Cells were grown to approximately 80% confluence and harvested by trypsinization. Cells were washed once with normal growth medium and twice with D-PBS. Homogenization, electrophoresis, and blotting procedures were as described previously (25) .

Recombinant PC3 Preparation

Monolayers of COS 7 cells in 15-cm dishes were infected with recombinant vaccinia virus VV:PC3 (5) at a multiplicity of infection = 10 and incubated overnight in normal growth medium. Cells were washed twice with warm D-PBS, then twice with Opti-MEM (Life Technologies, Inc.) containing 500 units/ml Trasylol, the pH of which had been adjusted to 6.0 with acetic acid. Cells were overlaid with 5 ml of this acidified medium and incubated at 30 °C for 3 h with occasional rocking. Media were collected, centrifuged for 10 min at 600 times g to remove any floating cells, and concentrated by ultrafiltration in Centriprep 30 (Amicon) at 4 °C, after addition of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM 1,10-phenanthroline, 10 µM E64, and 10 µM pepstatin). The pH was then adjusted to 5.5 with sodium acetate. After a 1-h incubation on ice, the PC3 preparation was centrifuged for 20 min at 15,000 times g, aliquoted, and stocked at -80 °C before use for in vitro conversion. The final protein concentration was about 1 mg/ml. Western blot analysis indicated that the major part of PC3 immunoreactive material migrated on SDS-PAGE with an apparent size of 87 kDa, with several other minor bands between 65 and 75 kDa (not shown). Active PC3 was also recovered from AtT-20 media using the same protocol.

In Vitro Conversion Studies

I-labeled GLP-1 and proinsulin were used as substrates for in vitro conversion studies performed with recombinant PC3. GLP-1-(1-36)-amide and GLP-1-(1-37) synthetic peptides were iodinated by the chloramine-T method. I-labeled human proinsulin was obtained from Eli Lilly. Based on modifications of other published procedures(28, 29) , reactions were done in 0.1 M sodium acetate buffer, pH 5.5, containing 0.1% Triton X-100, 10 µM dithiothreitol, 5 mM CaCl(2), and a mixture of protease inhibitors (10 µM E-64, 10 µM pepstatin, 0.2 mML-1-tosylamido-2-phenylethyl chloromethyl ketone, 0.1 mMN-p-tosyl-L-lysine chloromethyl ketone, and 10 µM guanidinoethylmercaptosuccinic acid). Up to 50 µg of total proteins of the recombinant PC3 preparation were used with 10,000-20,000 cpm of substrate in a total volume of 100 µl. Reactions were incubated for 16 h at 32 °C, stopped by boiling, concentrated, and analyzed by SDS-PAGE under reducing conditions (30) and autoradiography. I-Proinsulin was also digested with trypsin (0.1 µg/ml) in the presence or absence of carboxypeptidase B (10 µg/ml) for various times in order to obtain migration standards. The highly cross-linked gel used (16.5% T, 5% C) allowed the resolution of the following peptides after reduction of the disulfide bridges: intact proinsulin, B chain linked to C peptide (unresolved from the same peptide extended with Lys + Arg), C peptide linked to A chain, di-Arg-B chain, B chain, and A chain (not shown). The C peptide is not detected, because it is not labeled. Similarly, partial digestion of I-labeled GLP-1 by endoproteinase Arg-C (Boehringer Mannheim) yields a 3.4-kDa peptide, having the same migration as GLP-1-(7-36)-amide.


RESULTS

Proglucagon Processing in AtT-20 Cells

Proglucagon processing was studied in AtT-20 cells after infection with a recombinant vaccinia virus engineered to express preproglucagon. Peptides produced following proglucagon expression were identified by continuous metabolic labeling, immunoprecipitation, and electrophoresis in SDS-PAGE. Fig. 2shows the result of a typical experiment of proglucagon expression in AtT-20 cells. Immunoprecipitation was performed from the cell lysates, with the anti-glucagon monoclonal antibody glu 001 and the anti-GLP-1 antiserum 2135. These antisera recognize equally well N- and/or C-terminally extended forms of these peptides. Three major glucagon-containing peptides were detected, with apparent molecular masses in SDS-PAGE of 19, 9, and 4.5 kDa (Fig. 2). These peptides have been identified previously as proglucagon, glicentin, and oxyntomodulin, respectively, in alphaTC1-6 cells(17) . A minor component of 3.4 kDa was also detected that corresponds to the size of glucagon. On the other hand, GLP-1 antiserum immunoprecipitated only one major peptide of 3.4 kDa, besides the 19-kDa precursor (Fig. 2). After overexposure, two minor components of 8 kDa and of 4 kDa were also detected. The 8-kDa GLP-1 immunoreactive peptide has been identified as MPGF, a product of proglucagon processing in alpha cells, and the 4-kDa GLP-1 has the same electrophoretic mobility as the GLP-1-(1-37) and/or the GLP-1-(1-36)-amide(17) . The detection of these peptides in low amounts suggests their involvement as intermediates in the processing of proglucagon to the 3.4-kDa GLP-1 peptide. The apparent molecular size of this peptide corresponds closely to those calculated for bioactive GLP-1-(7-37) or GLP-1-(7-36)-amide.


Figure 2: Identification of the proglucagon-derived peptides produced in AtT-20 cells. AtT-20 cells were infected with VV:GLU (lanes 1 and 4) or the control VV:WT (lanes 2 and 3) virus, labeled with [^3H]Phe (lanes 1 and 2) or [S]Met (lanes 3 and 4) for 12 h. Immunoprecipitations were performed on cell lysates with antibodies directed against glucagon (lanes 3 and 4) or against GLP-1 (lanes 1 and 2). Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated. PRO, proglucagon; GLI, glicentin (proglucagon-(1-69)); OXT, oxyntomodulin (proglucagon 33-69); GLU, glucagon (proglucagon 33-61); MPGF, major proglucagon fragment (proglucagon-(72-158/160)); GLP-1, N-terminally extended glucagon-like peptide 1 (proglucagon-(72-108) and/or proglucagon-(72-107)-amide); tGLP-1, truncated glucagon-like peptide 1 (proglucagon-(78-108) and/or proglucagon(78-107)-amide).



The identification of MPGF as a processing intermediate was also supported by its detection in the conditioned medium of infected cells. In the absence of added secretagogue, infected cells secreted large amounts of proglucagon, glicentin, and MPGF (Fig. 3). Shorter peptides, that could be immunoprecipitated from the cell lysates, were not detected in the medium. It is a well established observation that AtT-20 cells and other transformed endocrine cells in culture constitutively secrete large amounts of unprocessed precursors and intermediates resulting from the earliest cleavage step(26) . Thus, the finding of secreted material cleaved at the interdomain Lys-Arg site suggests that this is the first cleavage during proglucagon processing in AtT-20 cells, as has also been demonstrated in alphaTC1-6 cells(17) . Following this cleavage, the N-terminal domain (glicentin) is only partially converted to oxyntomodulin, whereas the C-terminal domain (MPGF) is extensively processed to the 3.4-kDa GLP-1, probably via 4-kDa GLP-1 as an intermediate.


Figure 3: Identification of the proglucagon-derived peptides secreted by AtT-20 cells under unstimulated conditions. AtT-20 cells were infected and labeled as in Fig. 2. Immunoprecipitations were carried out on the media, with antibodies directed against glucagon (lanes 3 and 4) or against GLP-1 (lanes 1 and 2). Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated. Abbreviations are as in Fig. 2.



The putative 3.4-kDa GLP-1 was further characterized by radiosequencing of the [^3H]Phe-labeled peptide, purified by immunoprecipitation with the carboxyamidation-specific antiserum 89-390, followed by HPLC. Two peaks of radioactivity indicated the presence of Phe residues in positions 6 and 22 of the peptide (Fig. 4). This result demonstrates that the 3.4-kDa GLP-1 produced in AtT-20 cells is indeed GLP-1-(7-36)-amide or tGLP-1. This result also implies that a cleavage at the monobasic Arg site had occurred during the processing of proglucagon in AtT-20 cells, in addition to the other observed cleavages, that all occurred at dibasic sites.


Figure 4: Identification by radiosequencing of the tGLP-1 produced in AtT-20 cells. GLP-1 was immunoprecipitated with carboxyamidation specific antiserum 89-390 from a lysate of AtT-20 cells infected with VV:GLU and radiolabeled with [^3H]Phe. The immunoprecipitated material was further purified by reverse-phase HPLC and submitted to 30 cycles of Edman degradation. Anilinothiazolinone derivatives were collected at each cycle and assayed for radioactivity in a liquid scintillation counter. The sequence of proglucagon-(78-107) is shown under the graph.



Processing of Proglucagon in AtT-20/PC2 Cells

We have previously shown that PC2 processes proglucagon to glucagon in vivo, in alphaTC1-6 cells(17) . However, this conclusion was recently contested(31) . The low amounts of both glucagon production and PC2 expression in AtT-20 cells thus provided an opportunity to examine the effect of PC2 during co-expression experiments in these cells. As shown in Fig. 5, infection with VV:GLU of AtT-20 cells stably transfected with PC2 (AtT-20/PC2 cells) resulted in the accumulation of glucagon, whereas in a parallel experiment, wild type AtT-20 cells only accumulated glicentin and oxyntomodulin at detectable levels. This result demonstrates that PC2 is indeed able to process proglucagon to glucagon, or at the least glicentin to glucagon in this instance, as already shown in our earlier study with alphaTC1-6 cells.


Figure 5: Identification of the glucagon-containing peptides produced in AtT-20/PC2 cells. AtT-20/PC2 (lanes 1 and 2) or wild type AtT-20 cells (lanes 3 and 4) were infected with VV:GLU (lanes 2 and 4) or the control VV:WT (lanes 1 and 3) virus and labeled with [S]Met for 12 h. Glucagon-containing peptides were immunoprecipitated from the cell lysates and analyzed by SDS-PAGE. Abbreviations are as in Fig. 2.



Proglucagon Processing in STC-1 and betaTC-3 Cells

Since proglucagon was processed to glucagon in PC2 expressing alphaTC1-6 cells, and to tGLP-1 in PC3 expressing AtT-20 cells, we then investigated cells expressing both convertases. Immunoblot analysis of various cell lines revealed that both PC2 and PC3 were expressed in STC-1 and betaTC-3 cells, although at different levels (Fig. 6). STC-1, a mouse intestinal tumor cell line expressing proglucagon, was found to express PC2 and PC3 at moderate levels, whereas betaTC-3, a mouse insulinoma-derived cell line, expressed higher levels of both enzymes, as previously reported(32) . mRNA for both PC2 and PC3 were also detected in these two cell lines by RT-PCR (not shown).


Figure 6: Immunoblot analysis of the expression of PC2 and PC3 in four endocrine cell lines. 25 µg of proteins of a crude granular fraction of alphaTC1-6 (lanes 1 and 5), betaTC3 (lanes 2 and 6), AtT-20 (lanes 3 and 7), and STC1 cells (lanes 5 and 8) were resolved in SDS-PAGE and electrophoretically transferred onto an Immobilon P membrane. The blot was developed using the PC3 antiserum 2B6 (lanes 1-4) or PC2 antiserum PC2pep4 (lanes 5-8).



The processing of the N-terminal domain of the precursor in STC-1 cells was analyzed by immunoprecipitation with the glucagon antibody. Three major glucagon-containing peptides of 9, 7.5, and 3.4 kDa were found (Fig. 7). Based on their apparent molecular masses, and on their strong immunoreactivity toward the C-terminal specific glucagon antiserum P7 (not shown), the 3.4-kDa and 7.5-kDa peptides were identified as glucagon and proglucagon-(1-61) (GRPP linked to glucagon, see Fig. 1), respectively. The 9-kDa peptide was identified as the glicentin. A minor component of 4.5-kDa was also detected (Fig. 7) that was not immunoprecipitated with the P7 antiserum (not shown). This peptide was therefore identified as oxyntomodulin. These four glucagon-containing peptides have been identified previously in alphaTC1-6 cells(17) .


Figure 7: Proglucagon processing in STC1 cells. STC1 cells were labeled with [^3H]Phe for 12 h. Immunoprecipitations were performed on cell lysates with antibodies directed against glucagon (lanes 2) or against the C-terminal sequence of amidated GLP-1 (lane 1). Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated.



The processing of the C-terminal domain was analyzed by immunoprecipitation with GLP-1 antisera. The carboxyamidation-specific antiserum 89-390 immunoprecipitated a 4-kDa and a 3.4-kDa peptide, the latter being much more abundant than the former (Fig. 7). Similar experiments with the GLP-1 antiserum 2135 also immunoprecipitated these two peptides altogether with small amounts of the 19-kDa proglucagon and of the 8-kDa MPGF (not shown). On the other hand, antiserum 165-3, which is specific for the N-terminal extension of GLP-1-(1-37), immunoprecipitated the 19-, 8-, and 4-kDa GLP-1-containing peptides, but failed to react with the 3.4-kDa peptide (not shown). Taken together, these results suggest that the C-terminal domain of proglucagon is efficiently processed to tGLP-1, whereas the processing of the N-terminal domain to glucagon is incomplete in STC-1 cells, as previously shown by Blache et al.(33) .

The analysis of the processing of proglucagon expressed in betaTC-3 cells after infection with VV:GLU was complicated by two problems. First, infection of this cell line with the recombinant viral vector was found to result in a much lower expression of proglucagon than in the other cell lines used in this study. Second, very similar results were obtained after infection with VV:GLU and with the control wild type virus, at low multiplicity of infection (not shown). We reasoned from these observations that betaTC-3 cells are probably poorly infected by vaccinia virus and that this cell line is also endogenously expressing proglucagon at a low level, thus resulting in similar patterns of immunoprecipitated bands after infection with the recombinant VV:GLU or the control virus. Endogenous proglucagon expression in betaTC-3 cells was confirmed by RT-PCR (data not shown). Then, using a higher multiplicity of infection and a longer time of contact with the virus, a clear overexpression of proglucagon was observed in the VV:GLU infected cells compared to the control cells infected with the wild type virus, as shown in Fig. 8. Glucagon-containing peptides were immunoprecipitated from media and cell extracts after concentration on SepPak cartridges. The 19-kDa precursor and the 3.4-kDa glucagon were detected in the cells infected with VV:GLU, whereas only glucagon was immunoprecipitated from the cells infected with the control VV:WT. The higher amounts of glucagon detected after infection with the recombinant virus indicated that both the endogenously expressed proglucagon and the precursor produced by the viral vector are processed to glucagon. The accumulation of unprocessed precursor after recombinant vaccinia virus infection has often been noted by others(5, 34) . On the other hand, the apparent absence of intracellular accumulation of processing intermediates suggests that proglucagon is efficiently processed to glucagon in betaTC-3 cells. This apparent absence of intermediates could also reflect a pathway of processing different from the one observed in alphaTC1-6 cells, but this alternative is less likely, since a 9-kDa glicentin was detected in the medium (Fig. 8), suggesting that it is an intermediate in the processing of proglucagon to glucagon in betaTC-3 cells, as it is in alphaTC1-6 cells(17) . Similarly, only proglucagon and tGLP-1 sized peptides were detected after immunoprecipitation with GLP-1 antiserum (not shown). All these results indicate that betaTC-3 cells endogenously express proglucagon and process it both to glucagon and to tGLP-1. Thus, in contrast to alphaTC1-6 cells and AtT-20 cells, which express either PC2 or PC3 and process proglucagon either to glucagon or to tGLP-1, respectively, STC-1 and betaTC-3 cells, which express both PC2 and PC3, are able to process proglucagon to both glucagon and tGLP-1.


Figure 8: Proglucagon processing in betaTC3 cells. betaTC3 cells were infected with VV:GLU (lanes 2 and 4) or the control VV:WT (lanes 1 and 3) virus, labeled with [S]Met for 12 h. Immunoprecipitations were performed on cell lysates (lanes 3 and 4) and on medium (lanes 1 and 2) with antibody directed against glucagon. Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated. Abbreviations are as in Fig. 2.



Processing of Proglucagon in GH(4)C(1)and COS 7 Cells

Our results thus far supported the involvement of PC2 and PC3 in the processing of proglucagon to glucagon and tGLP-1, respectively. We then investigated its processing when expressed in cells lacking these two convertases. GH(4)C(1) cells, a rat pituitary tumor cell line known to express neither PC2 nor PC3(35) , and COS 7 cells, a monkey kidney fibroblast-like cell line, were used. Absence of any detectable level of PC2 and PC3 expression was confirmed at the RNA level by Northern blot and at the protein level by immunoblot analysis (data not shown).

Infection of GH(4)C(1) cells with VV:GLU resulted in the production of mainly unprocessed precursor (Fig. 9). The glucagon antibody immunoprecipitated large amounts of 19-kDa proglucagon and small amounts of 9-kDa glicentin, and the GLP-1 antiserum immunoprecipitated the precursor and small amounts of 8-kDa MPGF. Immunoprecipitations from the medium resulted in a very similar pattern (not shown). These results suggest that GH(4)C(1) cells, although able to store a fraction of the expressed proglucagon in an intracellular compartment, are unable to efficiently process it, as previously reported by Drucker et al.(36) . The only partial cleavage observed occurred at the interdomain Lys-Arg site and may have been due to the presence of low levels of PC2, PC3, or other proteases.


Figure 9: Proglucagon processing in GH(4)C(1) cells. GH(4)C(1) cells were infected with VV:GLU and labeled for 12 h. Immunoprecipitations were performed on cell lysates with antibodies directed against glucagon (lane 2) or against GLP-1 (lane 1). Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated.



The lack of processing was even more obvious with COS 7 cells. After infection with VV:GLU and radiolabeling, we were unable to immunoprecipitate any proglucagon or proglucagon-derived peptides from cell extracts, even after concentration on SepPak cartridge (not shown). However, immunoprecipitation from the medium revealed the presence of large amounts of proglucagon (Fig. 10). No smaller forms were detected even after prolonged exposure. These results indicate that COS 7 cells rapidly secrete proglucagon, without being able to process it at any of its potential cleavage sites, despite the presence of furin in these cells(37) .


Figure 10: Proglucagon processing in COS 7 cells. COS 7 cells were infected with the wild type virus (lane 1), or the VV:GLU, and labeled with [S]Met for 12 h. Media were concentrated and immunoprecipitated with the glucagon (lanes 1 and 2) or GLP-1 (lane 3) antibody.



The results of the various expression experiments in the cell lines are summarized in Table 1. A good correlation between PC2 expression and ability to process proglucagon to glucagon (efficient cleavage at both Lys-Arg and Lys-Arg sites) can be observed. Similarly, expression of PC3 correlates with processing to tGLP-1 (efficient cleavage at both Arg and Arg-Arg sites).



In Vitro Study of the Monobasic Cleavage

Since all the cells expressing PC3 were found able to process proglucagon to tGLP-1, we then asked whether PC3 can perform the cleavages necessary to release tGLP-1 from the precursor. This conversion involves cleavages of a dibasic site at the C terminus and of a monobasic site at the N terminus of the hormone sequence. PC3 has been shown previously to be able to cleave the dibasic site at the C terminus of GLP-1 in vitro, but the monobasic site cleavage has not been investigated (31) . It is well established that PC3 is able to process precursors at certain monobasic sites(34) . Consequently, we decided to test the ability of this convertase to cleave at the monobasic site occurring in the proglucagon sequence. In order to do this experiment, PC3 was expressed in COS 7 cells using a recombinant vaccinia virus. The medium was concentrated and used as crude enzyme preparation for incubations with radioiodinated substrates in vitro. A protease inhibitor mixture was included in order to inhibit any possible contaminating proteolytic activities. Incubation of this PC3 preparation with I-labeled human proinsulin indicated that it was active enough to process this substrate completely at the B chain-C peptide junction, and partially at the C peptide-A chain junction, as shown by the recovery, after reduction of the disulfide bridges, of three peptides having the same mobility in SDS-PAGE as the A chain of insulin, the B chain extended with two arginines, and the C peptide linked to the A chain (Fig. 11). Replacement of the calcium chloride in the incubation mixture by EDTA, or use of concentrated medium from VV:WT infected cells instead of the PC3 preparation, completely abolished the conversion of the proinsulin, indicating that the processing activity was indeed due to PC3. However, the same preparation was found to be unable to convert GLP-1 to tGLP-1 in parallel experiments (Fig. 11). The experiment was repeated several times with different preparations of recombinant PC3 using either I-GLP-1-(1-37) or I-GLP-1-(1-36)-amide as a substrate, with essentially the same results. Occasionally, a slight band having the apparent molecular mass of tGLP-1 and of the product generated by the endoproteinase Arg-C was observed (data not shown), but this observation was inconsistent and possibly came from traces of incompletely inactivated contaminating proteases. The same experiment was carried out with AtT-20 cell concentrated medium, and the same result was obtained (not shown). AtT-20 medium also contained a calcium-dependent proinsulin processing activity having enzymatic properties very similar to that of recombinant PC3. In contrast, no detectable processing of GLP-1 to tGLP-1 was observed, indicating that the monobasic cleaving endoprotease able to produce tGLP-1 in AtT-20 cells is either not secreted or is inactivated under the conditions used for these experiments. Moreover, the presence of large amounts of active PC3 in both AtT-20 conditioned medium and recombinant PC3 preparations suggests that PC3 is not the convertase cleaving the monobasic site and gives support to the hypothesis that an additional, as yet unidentified convertase may be required for proglucagon processing to tGLP-1 in AtT-20 cells and in cells with the L cell phenotype.


Figure 11: In vitro activity of recombinant PC3 on GLP-1 and proinsulin. I-labeled proinsulin (A) and GLP-1 (B) were used as substrates for in vitro conversion studies performed with recombinant PC3, obtained from concentrated medium of VV:PC3-infected COS 7 cells (lanes 3 and 6). Medium of VV:WT-infected COS 7 cells was used as a negative control (lanes 2 and 5). Incubations without enzyme are shown in lanes 1 and 4. Reactions were incubated for 16 h at 32 °C, stopped by boiling, concentrated, and analyzed by SDS-PAGE under reducing conditions and autoradiography. Similarly, digestion of I-labeled GLP-1 by endoproteinase Arg-C yield a 3.4-kDa peptide, having the same migration as GLP-1-(7-36)-amide (lane 7). Designations are: Pro, intact proinsulin; C-A, C-peptide-A chain fragment; B chain, B chain Arg-Arg; 1-36, GLP1-(1-36)-amide; 7-36, GLP1-(7-36)-amide.




DISCUSSION

We have examined the processing of proglucagon in AtT-20 cells after expression with a recombinant vaccinia virus vector. We have shown that this processing is closely similar to that observed in intestinal L cells in that the C-terminal domain is efficiently converted to tGLP-1 (12) while the N-terminal domain is only partially cleaved to oxyntomodulin and very low amounts of glucagon(19) . These results are in good agreement with the recent report by Mineo et al.(38) , who also found that transfected AtT-20 cells process proglucagon to oxyntomodulin to a much greater extent than to glucagon. This resemblance in processing patterns makes AtT-20 cells a good model for studying proglucagon processing in cells with the L cell phenotype and suggests that AtT-20 cells and intestinal L cells are endowed with similar endoproteolytic processing activities.

Of the six SPCs identified thus far, only PC2 and PC3 have been shown to be active in the regulated secretory pathway of neuroendocrine cells. In AtT-20 cells, PC3 is expressed at a much higher level than PC2(4) , whereas in alphaTC1-6 cells and pancreatic alpha cells, PC2 is present in large excess over PC3(17, 23, 24) . We have previously shown that PC2 is the key endoprotease responsible for proglucagon processing in cells with the alpha cell phenotype(17) . The results presented here further support this conclusion and make PC3 a good candidate for an endopeptidase involved in the processing of proglucagon in L cells. Indeed, we have shown that only PC3-expressing cell lines are able to efficiently process the C-terminal domain of the precursor to tGLP-1. This is further supported by the recent report by Rothenberg et al.(31) that PC3 is able to cleave in vitro the Lys-Arg, Lys-Arg, and Arg-Arg processing sites of proglucagon, cleavages that also occur in AtT-20 cells. Furthermore, Scopsi et al.(25) have detected immunoreactive PC3, but not PC2 in intestinal L cells. All these data are compatible with the hypothesis that PC3 is involved in the processing of proglucagon in L cells.

Just as PC3 expression is correlated with the ability of a cell line to process the C-terminal domain to tGLP-1, PC2 appears required for a cell line to be able to process the N-terminal domain to glucagon. Mineo et al.(38) also have shown that GH(3) and InR1-G9 cells, both expressing PC2, can convert proglucagon to glucagon. This confirms and extends our previous results with the alphaTC1-6 cell line(17) , suggesting that PC2 is the only SPC able to convert proglucagon to glucagon. However, Rothenberg et al.(31) have recently suggested, on the basis of the result of an in vitro conversion experiment, that PC2 by itself could not cleave the Lys-Arg processing site at the C terminus of the glucagon moiety. Accordingly, they concluded that PC2 would only be able to produce oxyntomodulin, and that a second endopeptidase, possibly PC6, would cleave at the C terminus of glucagon in alpha cells. Although this hypothesis can account for the observed lack of cleavage by PC2 in vitro, it is not compatible with results from in vivo processing studies. First, the involvement of PC6 can be ruled out, since betaTC3 cells which do not express detectable levels of PC6 (35) efficiently process proglucagon to glucagon. Likewise, the glucagon-producing alphaTC1-6 cells do not express detectable levels of PC6, as assessed by RT-PCR. (^2)Similar conclusions can be drawn for all the SPC presently known, except for PC2, which is the only convertase expressed in all the cell lines that are able to produce glucagon and only in those cell lines. Moreover, our results from expression of proglucagon in AtT-20/PC2 cells leave no doubt concerning the ability of PC2 to cleave the Lys-Arg processing site at the C terminus of the glucagon moiety in vivo. Perhaps the low activity of the PC2 preparation used by Rothenberg et al.(31) , which only partially cleaved the Lys-Arg and Lys-Arg sites, accounts for its failure to cleave this site in vitro. It is also possible that a factor present in vivo is missing in the in vitro experiment. On the other hand, some sites appear to be more ``resistant'' to cleavage, possibly due to a less favorable conformation, and, consequently, the level of expression of the convertase also has to be taken into consideration. For example, STC-1 cells that express lower levels of PC2 incompletely convert the N-terminal domain of proglucagon to glucagon. Similarly, limiting amounts of PC3 only partially convert proinsulin to des-31,32-proinsulin in vitro(39) , although higher amounts of PC3 are effective in converting this intermediate to insulin (see Fig. 11).

The differential processing of proglucagon underscores the very significant differences in site selectivity exhibited by PC2 and PC3, as already documented in studies with other precursors such as POMC (5, 6) and proinsulin(4) . Although it is now clear that both of these endoproteases most often cleave prohormones at dibasic sites, the precise structural basis for their site preferences on their physiological substrates is still unclear. Thus, in studies on proglucagon and proinsulin processing PC2 appears to prefer Lys-Arg sites while PC3 prefers Arg-Arg sites, but this is not the case with POMC processing, where various Lys-Arg sites are preferentially cleaved by either PC2 or PC3(5, 6) . Other sequence and/or conformational elements are thus likely to be crucial determinants of PC2 or PC3 cleavage motifs. A comparison of the amino acid sequences surrounding the sites preferred by PC2 or PC3 in proinsulin, proglucagon, and POMC does not reveal any distinguishing features in this small sample. Also, very little is known about the conformation of the polypeptide chain at cleavage sites. It seems possible that cleavage at one site in a precursor may generate conformational changes at other sites that in turn would then become better substrates for one or the other convertase, as suggested in the case of proinsulin(40) . This idea is further supported by our observations that in proglucagon cleavages do not occur randomly, but are temporally ordered. In alphaTC1-6 cells, PC2 cleaves first at Lys-Arg and then at both Lys-Arg and Lys-Arg sites in the glicentin produced by the first cleavage, but does not cleave these sites in intact proglucagon(17) . Similarly in AtT20 cells, PC3 cleaves first at the Lys-Arg site and then is able to further process the C-terminal domain.

Production of bioactive tGLP-1 involves the cleavage of the proglucagon at Arg, a monobasic processing site. Interestingly, PC3 has been reported recently to cleave prodynorphin at a monobasic site(34) . It has been suggested that the lack of a P2 basic residue is compensated by the presence of a P4 arginine(41) . However, the monobasic cleavage site in proglucagon has a glutamic residue at this position, a characteristic it shares with the monobasic processing site of prosomatostatin(42, 43) , which is cleaved inefficiently by PC3(44) . In contrast, transfected AtT-20 cells process prosomatostatin efficiently at this site(45, 46) . Our results indicate a similar discrepancy between the apparent lack of cleavage at the monobasic site in GLP1 by PC3 in vitro versus the high efficiency of tGLP-1 production from proglucagon in AtT-20 and other PC3-expressing cells. Thus, unless the in vitro activity of PC3 is not representative of its in vivo activity, this result suggests that an additional endopeptidase may be required in the regulated secretory pathway of AtT-20 cells (and intestinal L cells) to produce bioactive tGLP-1.

Potential candidates for this monobasic cleaving endoprotease include a putative mammalian homolog to the yeast YAP3-encoded aspartyl protease (47) . This enzyme has been shown to efficiently process anglerfish prosomatostatin II at its monobasic but not at its dibasic site, both in vitro and in vivo(48, 49) . Moreover, an aspartyl protease has been purified from anglerfish Brockmann bodies, that specifically cleaves this monobasic site(50) , suggesting that YAP3 homologs could be present in the endocrine cells of higher eukaryotes. A second candidate for a GLP-1 convertase is the dynorphin-converting enzyme, a thiol protease capable of cleaving Dyn B-29 to Dyn B-13 in vitro (a processing event occurring at a single arginine). This enzyme is present at high levels in the brain, the pituitary, and the ileum, and might also be involved in monobasic processing of precursors other than prodynorphin(51) . Finally, PACE4 has been reported recently to cleave prosomatostatin at its monobasic site in the constitutive secretory pathway(44) , raising the possibility that an isoform or homolog of this enzyme could have the same specificity in the regulated pathway, where the monobasic cleavage of proglucagon is thought to occur. Interestingly, four isoforms of PACE4 produced by alternative splicing of the mRNA have so far been cloned(52, 53) . One of these isoforms, PACE4C, is probably functional and is expressed in the beta cells, but not in the alpha cells of the islets of Langerhans, as shown by immunocytochemistry(23) . However, it is not known whether this enzyme enters the regulated secretory pathway.

In conclusion, this study confirms that PC2 is the convertase responsible for processing proglucagon to glucagon in the alpha cell and provides evidence that PC3 is very likely to be the convertase involved in the processing of proglucagon in L cells. However, the monobasic cleavage necessary to release the bioactive tGLP-1 is not efficiently performed by PC3 in vitro, suggesting that an as yet unidentified additional convertase may also be required to achieve the characteristic pattern of proglucagon processing of the intestinal L cell. Further investigation of the susceptibility of the tGLP-1 production to various protease inhibitors, both in vivo and in vitro in secretory granule lysates, may lead to a more thorough characterization of the convertase involved in this monobasic cleavage.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants DK 13914 and DK 20595 and in part by the Howard Hughes Medical Institute. 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.

§
Present address: Laboratoires de Recherche Louis Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.

To whom correspondence should be addressed: The Howard Hughes Medical Institute, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-1334; Fax: 312-702-4292.

(^1)
The abbreviations used are: POMC, proopiomelanocortin; BSA, bovine serum albumin; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GLP, glucagon-like peptide; tGLP, truncated GLP-1 (GLP1-(7-37)); GRPP, glicentin-related polypeptide; HPLC, high performance liquid chromatography; IP, intervening peptide; MPGF, major proglucagon fragment; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction; SPC, subtilisin-like proprotein convertase; VV, vaccinia virus.

(^2)
Q. Gong, unpublished data.


ACKNOWLEDGEMENTS

We thank Terri Reid for assistance with cell cultures and maintenance of cell lines and Florence Rozenfeld for expert assistance in the preparation of this manuscript.


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