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• W2096728414 abstract "Proglucagon is processed differently in the islet α 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 α cell line (αTC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical α 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 α cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and βTC-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, GH4C1 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 GH4C1 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. Proglucagon is processed differently in the islet α 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 α cell line (αTC1-6) we demonstrated that the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is responsible for generating the typical α 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 α cell processing phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and βTC-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, GH4C1 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 GH4C1 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. INTRODUCTIONThe 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(1Steiner D.F. Smeekens S.P. Ohagi S. Chan S.J. J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar, 2Van de Ven W.J.M. Roebroek A.J.M. Van Duijnhoven H.L.P. Crit. Rev. Oncogenesis. 1993; 4: 115-136PubMed Google Scholar, 3Seidah N.G. Chrétien M. Trends Endocrinol. Metab. 1992; 3: 133-140Abstract Full Text PDF PubMed Scopus (157) Google Scholar). 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(1Steiner D.F. Smeekens S.P. Ohagi S. Chan S.J. J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar, 3Seidah N.G. Chrétien M. Trends Endocrinol. Metab. 1992; 3: 133-140Abstract Full Text PDF PubMed Scopus (157) Google Scholar). PC2 and PC3 both participate in the processing of proinsulin to insulin in the pancreatic β cell (4Smeekens S.P. Montag A.G. Thomas G. Albiges-Rizo C. Carroll R. Benig M. Phillips L.A. Martin S. Ohagi S. Gardner P. Swift H.H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8822-8826Crossref PubMed Scopus (257) Google Scholar) and of POMC 1The abbreviations used are: POMCproopiomelanocortinBSAbovine serum albuminD-PBSDulbecco's phosphate-buffered salineDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumGLPglucagon-like peptidetGLPtruncated GLP-1 (GLP1-(7-37))GRPPglicentin-related polypeptideHPLChigh performance liquid chromatographyIPintervening peptideMPGFmajor proglucagon fragmentPAGEpolyacrylamide gel electrophoresisRT-PCRreverse transcription-polymerase chain reactionSPCsubtilisin-like proprotein convertaseVVvaccinia virus. to ACTH and/or other products in the pituitary(5Thomas L. Leduc R. Thorne B.A. Smeekens S.P. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5297-5301Crossref PubMed Scopus (275) Google Scholar, 6Benjannet S. Rondeau N. Day R. Chrétien M. Seidah N.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3564-3568Crossref PubMed Scopus (535) Google Scholar). Moreover, it has been demonstrated that the known differential processing of POMC to different products in the anterior versus intermediate lobes of the pituitary (7Eipper B.A. Mains R.E. Endocrine Rev. 1980; 1: 1-27Crossref PubMed Scopus (811) Google Scholar) is due to the differential expression of PC2 and PC3 in these lobes (8Day R. Schafer M.K.-H. Watson S.J. Chrétien M. Seidah N.G. Mol. Endocrinol. 1992; 6: 485-497Crossref PubMed Scopus (173) Google Scholar). 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 α cells of the islets of Langerhans in the pancreas and in the endocrine L cells of the intestinal mucosa(9Mojsov S. Heinrich G. Wilson I.B. Ravazzola M. Orci L. Habener J.F. J. Biol. Chem. 1986; 261: 11880-11889Abstract Full Text PDF PubMed Google Scholar, 10Novak U. Wilks A. Buell G. McEwen S. Eur. J. Biochem. 1987; 164: 553-558Crossref PubMed Scopus (103) Google Scholar); however, differential processing results in the formation of different sets of peptides with opposing biological activities. The pancreatic α 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(11Mojsov S. Weir G.C. Habener J.F. J. Clin. Invest. 1987; 79: 616-619Crossref PubMed Scopus (683) Google Scholar, 12Ørskov C. Bersani M. Johnsen A.H. H⊘jrup P. Holst J.J. J. Biol. Chem. 1989; 264: 12826-12829Abstract Full Text PDF PubMed Google Scholar).The pancreatic α 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(13Patzelt C. Schiltz E. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5007-5011Crossref PubMed Scopus (90) Google Scholar, 14Patzelt C. Tager H.S. Carroll R.J. Steiner D.F. Nature. 1979; 282: 260-266Crossref PubMed Scopus (96) Google Scholar). A small fraction (10 to 20%) of the MPGF is further processed to GLP-1(15Holst J.J. Bersani M. Johnsen A.H. Kofod H. Hartmann B. Ørskov C. J. Biol. Chem. 1994; 269: 18827-18833Abstract Full Text PDF PubMed Google Scholar). Low amounts of glicentin (proglucagon-(1-69)) have also been detected in the pancreas (16Sheikh S.P. Baldissera F.G.A. Karlsen F.Ø. Holst J.J. FEBS Lett. 1985; 179: 1-6Crossref PubMed Scopus (9) Google Scholar), suggesting that it is an intermediate in the formation of glucagon. This processing pathway has been demonstrated recently through pulse-chase experiments in αTC1-6 cells, an islet-derived cell line transformed with SV40 large T antigen(17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). Proglucagon is initially cleaved at the Lys79-Arg80 site to produce glicentin and MPGF. Glicentin is later processed at the Lys31-Arg32 and Lys62-Arg63 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 Arg109-Arg110 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(18Ørskov C. Holst J.J. Knuhtsen S. Baldissera F.G. Poulsen S.S. Nielsen O.V. Endocrinology. 1986; 119: 1467-1475Crossref PubMed Scopus (410) Google Scholar). Glucagon levels are very low in the intestine(19Kervran A. Blache P. Bataille D. Endocrinology. 1987; 121: 704-713Crossref PubMed Scopus (71) Google Scholar). Rather, the N-terminal domain remains incompletely processed in the form of glicentin (20Thim L. Moody A.J. Peptides. 1982; 2: 37-39Google Scholar) and is only partially cleaved into GRPP and oxyntomodulin (proglucagon-(33-69))(21Bataille D. Tatemoto K. Gespach C. Jörnvall H. Rosselin G. Mutt V. FEBS Lett. 1982; 146: 79-86Crossref PubMed Scopus (128) Google Scholar), a form of glucagon C-terminally extended with IP-1(22Tager H.S. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2321-2325Crossref PubMed Scopus (108) Google Scholar). An additional cleavage occurs at a single arginine residue within the GLP-1 sequence (Arg109), 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(12Ørskov C. Bersani M. Johnsen A.H. H⊘jrup P. Holst J.J. J. Biol. Chem. 1989; 264: 12826-12829Abstract Full Text PDF PubMed Google Scholar). 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 α and L cells. This view is supported by recent findings that pancreatic α cells express high levels of PC2 and low levels, if any, of PC3(17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar, 23Nagamune H. Muramatsu K. Akamatsu T. Tamai Y. Izumi K. Tsuji A. Matsuda Y. Endocrinology. 1995; 136: 357-360Crossref PubMed Scopus (38) Google Scholar, 24Neerman-Arbez M. Cirulli V. Halban P.A. Biochem. J. 1994; 300: 57-61Crossref PubMed Scopus (47) Google Scholar), whereas intestinal L cells contain immunoreactive PC3 but not PC2(25Scopsi L. Gullo M. Rilke F. Martin R. Steiner D.F. J. Clin. Endocrinol. Metab. 1995; 80: 294-301Crossref PubMed Google Scholar). 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 METHODSAntiseraGlu 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 (17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). 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 CultureAtT-20 cells and COS 7 cells were grown in DMEM supplemented with 10% FBS. AtT-20/PC2 cells (26Zhou A. Bloomquist B.T. Mains R.E. J. Biol. Chem. 1993; 268: 1763-1769Abstract Full Text PDF PubMed Google Scholar) 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.). βTC3 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. GH4C1 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% CO2, 95% air mixture in a humidified incubator.Vaccinia VirusIn 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(27Mackett M. Smith G.L. Moss B. Glover D.M. DNA Cloning, A Practical Approach. II. IRL Press, Washington, D. C.1985: 191-211Google Scholar). 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 GH4C1 cells) in 6-cm plates. Infections were carried out at a multiplicity of infection = 1 (10 for βTC3 cells) in 0.5 ml of DMEM at 37°C for about 30 min (90 min for βTC3 cells). Cells were returned to their normal growth medium for 6 h before metabolic labeling.Metabolic LabelingCells 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 35S or 3H (0.5 mCi per dish; [35S]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 (17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). Media were collected, centrifuged at 15,000 × 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 KH2PO4, 8.1 mM Na2HPO4), scraped off the plate, pelleted, and resuspended in 0.5 ml of immunoprecipitation buffer (50 mM Na2HPO4, 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 × g. The supernatants were used for immunoprecipitation.For some experiments with βTC3 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 × g to remove insoluble materials. The whole supernatant was used for immunoprecipitation. Control experiments with αTC1-6 cells showed a similar recovery of the glucagon-containing peptides with both extraction methods (not shown). Recovery of 125I-labeled glucagon by these methods was greater than 80%.ImmunoprecipitationsImmunoprecipitations 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%β-mercaptoethanol, pH 6.8), and the supernatant was analyzed by SDS-PAGE (28Rufaut N.W. Brennan S.O. Hakes D.J. Dixon J.E. Birch N.P. J. Biol. Chem. 1993; 268: 20291-20298Abstract Full Text PDF PubMed Google Scholar) and fluorography.RadiosequencingGLP-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 C18 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 AnalysisCells 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 (25Scopsi L. Gullo M. Rilke F. Martin R. Steiner D.F. J. Clin. Endocrinol. Metab. 1995; 80: 294-301Crossref PubMed Google Scholar).Recombinant PC3 PreparationMonolayers of COS 7 cells in 15-cm dishes were infected with recombinant vaccinia virus VV:PC3 (5Thomas L. Leduc R. Thorne B.A. Smeekens S.P. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5297-5301Crossref PubMed Scopus (275) Google Scholar) 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 × 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 × 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 Studies125I-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. 125I-labeled human proinsulin was obtained from Eli Lilly. Based on modifications of other published procedures(28Rufaut N.W. Brennan S.O. Hakes D.J. Dixon J.E. Birch N.P. J. Biol. Chem. 1993; 268: 20291-20298Abstract Full Text PDF PubMed Google Scholar, 29Zhou Y. Lindberg I. J. Biol. Chem. 1994; 269: 18408-18413Abstract Full Text PDF PubMed Google Scholar), reactions were done in 0.1 M sodium acetate buffer, pH 5.5, containing 0.1% Triton X-100, 10 μM dithiothreitol, 5 mM CaCl2, and a mixture of protease inhibitors (10 μM E-64, 10 μM pepstatin, 0.2 mML-1-tosylamido-2-phenylethyl chloromethyl ketone, 0.1 mMNα-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 (30Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10442) Google Scholar) and autoradiography. 125I-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 125I-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.RESULTSProglucagon Processing in AtT-20 CellsProglucagon 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. 2 shows 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 αTC1-6 cells(17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). 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 α 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(17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). 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 2Identification 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 [35S]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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)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(26Zhou A. Bloomquist B.T. Mains R.E. J. Biol. Chem. 1993; 268: 1763-1769Abstract Full Text PDF PubMed Google Scholar). Thus, the finding of secreted material cleaved at the interdomain Lys79-Arg80 site suggests that this is the first cleavage during proglucagon processing in AtT-20 cells, as has also been demonstrated in αTC1-6 cells(17Rouillé Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Crossref PubMed Scopus (178) Google Scholar). Following this cleavage, the N-" @default.
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• W2096728414 title "Differential Processing of Proglucagon by the Subtilisin-like Prohormone Convertases PC2 and PC3 to Generate either Glucagon or Glucagon-like Peptide" @default.
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