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• W2000917954 abstract "The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic α-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel α-cell line (αTC-ΔPC2) derived from PC2 homozygous null animals. The αTC-ΔPC2 cells are shown to be similar to the well characterized αTC1–6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in αTC-ΔPC2 leads to a complete block in the production of mature glucagon. Surprisingly, αTC-ΔPC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70–71). Further analysis reveals that αTC-ΔPC2 cells, unlike αTC1–6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that αTC-ΔPC2 cells provide additional evidence for PC2 as the major convertase in α-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases. The endoproteolytic processing of proproteins in the secretory pathway depends on the expression of selected members of a family of subtilisin-like endoproteases known as the prohormone convertases (PCs). The main PC family members expressed in mammalian neuroendocrine cells are PC2 and PC1/3. The differential processing of proglucagon in pancreatic α-cells and intestinal L cells leads to production of distinct hormonal products with opposing physiological effects from the same precursor. Here we describe the establishment and characterization of a novel α-cell line (αTC-ΔPC2) derived from PC2 homozygous null animals. The αTC-ΔPC2 cells are shown to be similar to the well characterized αTC1–6 cell line in both morphology and overall gene expression. However, the absence of PC2 activity in αTC-ΔPC2 leads to a complete block in the production of mature glucagon. Surprisingly, αTC-ΔPC2 cells are able to efficiently cleave the interdomain site in proglucagon (KR 70–71). Further analysis reveals that αTC-ΔPC2 cells, unlike αTC1–6 cells, express low levels of PC1/3 that lead to the generation of glicentin as well as low amounts of oxyntomodulin, GLP-1, truncated GLP-1, and N-terminally extended GLP-2. We conclude that αTC-ΔPC2 cells provide additional evidence for PC2 as the major convertase in α-cells leading to mature glucagon production and provide a robust model for further analysis of the mechanisms of proprotein processing by the prohormone convertases. Since the discovery of propeptide processing in the production of mature insulin (1Steiner D.F. Cunningham D. Spigelman L. Aten B. Science. 1967; 157: 697-700Google Scholar), endoproteolytic cleavage of cellular proteins to generate biologically active products has become recognized as a widespread and fundamental regulatory mechanism of the proteome. This type of processing occurs in multiple subcellular compartments, including the cytoplasm, various organelles, and on membrane surfaces (2Wilkinson K.D. Annu. Rev. Nutr. 1995; 15: 161-189Google Scholar, 3Nakayama K. Biochem. J. 1997; 327: 625-635Google Scholar, 4Martoglio B. Dobberstein B. Trends Cell Biol. 1998; 8: 410-415Google Scholar, 5Sakai J. Rawson R.B. Espenshade P.J. Cheng D. Seegmiller A.C. Goldstein J.L. Brown M.S. Mol. Cell. 1998; 2: 505-514Google Scholar, 6Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Google Scholar). Consequent to initial findings that proinsulin processing occurs in the regulated secretory pathway, multiple small peptide hormones and neuropeptides are now recognized as being derived from larger precursors by similar processing in post-Golgi compartments (7Steiner D.F. Kemmler W. Tager H.S. Peterson J.D. Fed. Proc. 1974; 33: 2105-2115Google Scholar, 8Seidah N.G. Chretien M. Day R. Biochimie (Paris). 1994; 76: 197-209Google Scholar, 9Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Google Scholar, 10Creemers J.W. Jackson R.S. Hutton J.C. Semin. Cell Dev. Biol. 1998; 9: 3-10Google Scholar, 11Seidah N.G. Day R. Marcinkiewicz M. Chretien M. Ann. N. Y. Acad. Sci. 1998; 839: 9-24Google Scholar, 12Viale A. Ortola C. Hervieu G. Furuta M. Barbero P. Steiner D.F. Seidah N.G. Nahon J.L. J. Biol. Chem. 1999; 274: 6536-6545Google Scholar, 13Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Google Scholar). Early studies of these processing events led to the recognition that peptide cleavage usually occurs C-terminal to dibasic amino acid motifs, followed by removal of the C-terminal basic amino acids by carboxypeptidases, such as carboxypeptidase E (14Fricker L. Annu. Rev. Physiol. 1988; 50: 309-321Google Scholar). Following the discovery of the yeast calcium-dependent subtilisin-like endoprotease kexin (Kex2p), which acts on dibasic motifs in the α mating factor and killer toxin precursors in the distal yeast secretory pathway (15Julius D. Brake A. Blair L. Kunisawa R. Thorner J. Cell. 1984; 37: 1075-1089Google Scholar, 16Fuller R.S. Sterne R.E. Thorner J. Annu. Rev. Physiol. 1988; 50: 345-362Google Scholar, 17Fuller R.S. Brake A. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1434-1438Google Scholar), the cloning and characterization of related mammalian enzymes soon followed (18Smeekens S.P. Steiner D.F. J. Biol. Chem. 1990; 265: 2997-3000Google Scholar, 19Smeekens S.P. Avruch A.S. LaMendola J. Chan S.J. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 340-344Google Scholar, 20Seidah N.G. Marcinkiewicz M. Benjannet S. Gaspar L. Beaubien G. Mattei M.G. Lazure C. Mbikay M. Chretien M. Mol. Endocrinol. 1991; 5: 111-122Google Scholar). These proteases have been designated subtilisin-like prohormone or proprotein convertases (SPCs, or more simply PCs) 1The abbreviations used are: PC, prohormone convertase; GLP-1, -2, glucagon-like peptides 1 and 2; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RT, reverse transcription; MCA, 7-methoxycoumarin; MOPS, 4-morpholinepropanesulfonic acid; SSPE, saline/sodium phosphate/EDTA. 1The abbreviations used are: PC, prohormone convertase; GLP-1, -2, glucagon-like peptides 1 and 2; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RT, reverse transcription; MCA, 7-methoxycoumarin; MOPS, 4-morpholinepropanesulfonic acid; SSPE, saline/sodium phosphate/EDTA. in recognition of their role in processing, not only prohormones, but a wide variety of other precursor proteins that traverse the secretory pathway. Although the mammalian PC family is now quite large, consisting of seven members with varying basic residue specificities, those mainly responsible for precursor processing in the regulated secretory pathway are now believed to be PC3 (also called PC1, and here referred to as PC1/3), PC2 (13Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Google Scholar), and to a lesser extent PC5/6A (21Barbero P. Rovere C. De Bie I. Seidah N. Beaudet A. Kitabgi P. J. Biol. Chem. 1998; 273: 25339-25346Google Scholar, 22Cain B.M. Vishnuvardhan D. Beinfeld M.C. Peptides. 2001; 22: 1271-1277Google Scholar, 23Dey A. Norrbom C. Xhu X. Stein J. Zhang C. Ueda K. Steiner D.F. Endocrinology. 2004; 145: 1961-1971Google Scholar). The PCs are all calcium-dependent serine endoproteases with acidic pH optima consistent with their function in the calcium-rich acidic secretory granules of neuroendocrine cells. A recent solution of the crystal structure of the PC furin confirms their structural relationship to the subtilase family and provides more detailed insight into the structural basis of substrate recognition (24Henrich S. Cameron A. Bourenkov G.P. Kiefersauer R. Huber R. Lindberg I. Bode W. Than M.E. Nat. Struct. Biol. 2003; 10: 520-526Google Scholar). Like subtilisin, these proteases become active by autocatalytic cleavage of an N-terminal propeptide, which is required for folding of the proenzymes (10Creemers J.W. Jackson R.S. Hutton J.C. Semin. Cell Dev. Biol. 1998; 9: 3-10Google Scholar, 25Hu Z. Zhu X. Jordan F. Inouye M. Biochemistry. 1994; 33: 562-569Google Scholar). A downstream domain of about 150 amino acids, called the P- or Homo B-domain (8Seidah N.G. Chretien M. Day R. Biochimie (Paris). 1994; 76: 197-209Google Scholar, 9Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Google Scholar), is also required for folding and activity. This domain plays a regulatory role, influencing both the calcium dependence and pH optima (26Zhou A. Martin S. Lipkind G. LaMendola J. Steiner D.F. J. Biol. Chem. 1998; 273: 11107-11114Google Scholar). The variable C-terminal regions of the PCs are less conserved and play a role in their subcellular localization (10Creemers J.W. Jackson R.S. Hutton J.C. Semin. Cell Dev. Biol. 1998; 9: 3-10Google Scholar, 27Steiner D.F. Curr. Opin. Chem. Biol. 1998; 2: 31-39Google Scholar). Although PC1/3 and PC2 share many biochemical and functional characteristics, subtle structural differences must account for observed differences in their recognition of distinct dibasic amino acid motifs within precursors (8Seidah N.G. Chretien M. Day R. Biochimie (Paris). 1994; 76: 197-209Google Scholar, 9Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Google Scholar, 13Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Google Scholar). This functional refinement between PC1/3 and PC2 becomes strikingly apparent in the processing of proglucagon, a multifunctional precursor that contains multiple cleavage sites recognized with varying efficiencies by these two convertases (28Rouille Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Google Scholar, 29Rouille Y. Martin S. Steiner D.F. J. Biol. Chem. 1995; 270: 26488-26496Google Scholar, 30Rouillé Y. Bianchi M. Irminger J.-C. Halban P. FEBS Lett. 1997; 413: 119-123Google Scholar, 31Rouillé Y. Kantengwa S. Irminger J.-C. Halban P.A. J. Biol. Chem. 1997; 272: 32810-32816Google Scholar). In this way proglucagon can be processed in different cells to yield unique mixtures of products with differing functions. Thus, in the pancreatic α-cell abundant expression of PC2 is associated with the nearly exclusive production of glucagon (28Rouille Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Google Scholar, 30Rouillé Y. Bianchi M. Irminger J.-C. Halban P. FEBS Lett. 1997; 413: 119-123Google Scholar), whereas in intestinal L cells processing, mediated mainly by PC1/3, leads instead to the production of glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) (29Rouille Y. Martin S. Steiner D.F. J. Biol. Chem. 1995; 270: 26488-26496Google Scholar, 31Rouillé Y. Kantengwa S. Irminger J.-C. Halban P.A. J. Biol. Chem. 1997; 272: 32810-32816Google Scholar). We report here the development and characterization of a transformed pancreatic α-cell line from PC2 null mice (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar), which has provided the opportunity to study proglucagon processing in a pure population of α-cells in vitro in the absence of PC2 activity. Reagents—The human 7B2 CT peptide, 1–31 amino acids, was a kind gift from Dr. Iris Lindberg of Louisiana State University Medical Center, New Orleans, LA. The rabbit anti GLP-1 antibody was a gift from Dr. Jens J. Holst of the Panum Institute, University of Copenhagen, Denmark. All the cell culture reagents and superscript first strand synthesis system for RT-PCR were purchased from Invitrogen. The TaqDNA polymerase and protein A-agarose were from Roche Diagnostics. The RNeasy kit for total RNA extraction was purchased from Qiagen. The Prime-It II kit, sonicated salmon sperm DNA, and Duralon-UV membrane were from Stratagene. The fluorogenic substrate, pyrGlu-Arg-Thr-Lys-Arg-MCA, and polyclonal antibody to glucagon were purchased from Peninsula Laboratories. The ECL Plus kit for Western blotting and the radioisotopes, α[32P]dCTP (3000 Ci/mmol) and [35S]-Met, were from Amersham Biosciences. Establishing the αTC-ΔPC2 Cell Line—Previously described PC2 heterozygous null mice (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar) were bred to Glu-TAg animals expressing the SV-40 large T antigen in the pancreatic islet α-cells (Glu2-TAg6 animals provided by Doug Hanahan, University of California at San Francisco) (33Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-4143Google Scholar). The resulting PC2 heterozygous null/Glu-TAg-positive animals were bred to PC2 homozygous null animals to produce PC2 homozygous null/Glu-TAg-positive animals. Genotyping was carried out by PCR analysis as previously described (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar, 33Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-4143Google Scholar). These animals were sacrificed at 6–7 months of age, and the induced glucagonomas, identified as solid tumor masses in pancreata measuring between 2 and 5 mm in diameter, were dissected and used to establish α-cell lines (33Powers A.C. Efrat S. Mojsov S. Spector D. Habener J.F. Hanahan D. Diabetes. 1990; 39: 406-4143Google Scholar). Tumors were minced with sterile scalpels and plated in high glucose Dulbecco's modified Eagle's medium, 2× Pen/Strep, and 10% fetal bovine serum. Once a week one-half of the medium was removed and replaced with fresh medium. Two weeks into culturing, thimerosal (Sigma) was added to 2 μg/ml for 3 days to deplete cultures of fibroblasts. Cells were passaged every 3–4 weeks, as needed using trypsin/EDTA. After 5 months of growth ∼two-thirds of the cells died abruptly. The remaining cells continued to grow well and were passaged for 3 more months. At this point (passage 12) cells were frozen in freezing media for long term storage and subsequent characterization. The (αTC-ΔPC2) cells described here were derived from a single tumor. Imaging—Cells were grown on glass coverslips and imaged on an Olympus BH-2 light microscope or in plastic culture flasks followed by processing for immunoelectron microscopy as previously described (34Webb G.C. Akbar M.S. Zhao C. Swift H.H. Steiner D.F. Diabetes. 2002; 51: 398-405Google Scholar). Cell Culture, Metabolic Labeling, Immunoprecipitation, and Western Blotting—αTC1–6, αTC-ΔPC2, βTC-3, AtT20, NIH-3T3, and COS-7 cells (1–2 × 106/60-mm dish) were cultured in Dulbecco's modified Eagle's medium (plus high glucose) supplemented with 2 mml-glutamine, 1 mm sodium pyruvate, penicillin (50 units/ml)-streptomycin (50 μg/ml) and 10% fetal bovine serum. Metabolic labeling, immunoprecipitation, and Western blotting were performed as described before (35Dey A. Xhu X. Carroll R. Turck C.W. Stein J. Steiner D.F. J. Biol. Chem. 2003; 278: 15007-15014Google Scholar). Aprotinin (5 μg/ml) was added in chase medium containing 10× excess cold methionine following metabolic pulse labeling. For immunoprecipitation of GLP-1 and GLP-2 peptides, dipeptidyl peptidase IV inhibitor (Linco Research) was added in both cell extracts and media. Total cell extracts (12,000 × g supernatants) as well as immunoprecipitated peptides from cell extracts and media were resolved in gradient Tricine/SDS-PAGE and regular SDS-PAGE under the reducing conditions using 5% β-mercaptoethanol in Laemmli sample buffer. RT-PCR—Total RNA was extracted from each cell type (1–2 × 106 cells/60-mm dish) as well as mouse pancreatic islets (two animals each for wild-type and knock out and of 12 weeks of age) using Qiagen's protocol. To synthesize first strand cDNA, 1 μg of total RNA for each sample and oligo(dT) supplied with the kit were used following Invitrogen's protocol. Amplification of each of the target cDNAs was achieved by PCR (1 hold at 94 °C for 3 min followed by 25 cycles with 1 min each at 94 °C, 55 °C, and 72 °C, respectively, and finally 1 hold at 72 °C for 5 min) using specific sense and antisense oligonucleotide primers for each gene. For amplification of the catalytically inactive PC2 cDNA, the identical primers used for the wild type PC2 were employed because these primers were designed to exclude the exon 3 region that was deleted to create PC2 knock out mice. All the PCR products were finally size-fractionated in 1% agarose gels and visualized and photographed under UV using a Polaroid MP-4 camera (Fotodyne Inc.). The forward (F) and reverse (R) oligonucleotide primers were: 1) PC2: F, CAT CAC AGT CAA CGC GAC CAG and R, TTT CTC AGG ATA CTT TGC AGG; 2) proglucagon: F, CTT CAA GAC ACA GAG GAG AAC C and R, CGC AGA GAT GTT GTG AAG ATG G; 3) proinsulin 1: F, CAA GTG GAA CAA CTG GAG CTG G and R, TAT TCA TTG CAG AGG GGT GGG G; and 4) proinsulin 2: F, AGT GGC ACA ACT GGA GCT GG and R, TAT TCA TTG CAG AGG GGT AGG C. PC2 Enzyme Assay—A 1-ml aliquot of 3-day-old cell culture medium (with serum) in duplicate/triplicate from each cell type having passage numbers varying from 15 to 20 was concentrated 20-fold using Microcon centrifugal filters (Amicon). Each concentrated aliquot was added in a previously reported assay mixture of 100 μl (final volume) containing the fluorogenic substrate, pyrGlu-Arg-Thr-Lys-Arg-MCA (36Cameron A. Apletalina E.V. Lindberg I. Dalbey R.E. Sigman D.S. Third Ed. The Enzymes. XXII. Academic Press, New York2002: 291-332Google Scholar), in the absence or presence of the PC2 inhibitory peptide, human 7B2 CT peptide (1–31 amino acids) (37Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Google Scholar), at a final concentration of 10 μm. The assay mixture was incubated for 18 h at 37 °C, and the amount of MCA liberated from the substrate as a result of enzymatic cleavage in each sample was measured following quenching with 1 m acetic acid as described before (37Zhu X. Rouille Y. Lamango N.S. Steiner D.F. Lindberg I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4919-4924Google Scholar). Northern Blotting—Total RNA from each cell type was extracted following Qiagen's protocol. Equal amounts of total RNA were size-fractionated in 1% agarose gels containing 0.2 m MOPS (pH 7.0) and 2.2 m formaldehyde and transferred onto Duralon membrane. It was baked at 80 °C for 2 h under vacuum to immobilize RNA. Membrane was pre-hybridized at 42 °Cfor 2 h in a solution containing 50% formamide, 6× SSPE, 0.5% SDS, 5× Denhardt's solution and 0.1 mg/ml sonicated salmon sperm DNA and hybridized in the same solution overnight at 42 °C in the presence of radiolabeled, denatured cDNA probe (specific activity, 108 dpm/μg) in a hybridization oven. Membrane was washed in 2× SSC at 50 °C for 30 min (2 × 15 min) followed by 0.2× SSC plus 0.1% SDS at 60 °C for 45 min (3 × 15 min) before exposing to film. In some cases, more stringent washes were performed using 0.1× SSC plus 0.1% SDS at 65 °C. For removal of hybridization probe to re-probe with a different cDNA, membrane was placed in a boiling stripping solution of 0.1× SSPE with 0.5% SDS for at least 10 min and rinsed with 1× SSPE before prehybridization. The cDNAs, used as hybridization probes, were synthesized by RT-PCR using specific sense and antisense primers for each gene. Microarray Analysis—αTC1–6 and αTC-ΔPC2 cells were processed for microarray analysis as described previously (38Wang J. Webb G. Cao Y. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12660-12665Google Scholar). Affymetrix set 430 microarrays, which assess over 34,000 mouse genes, were used. Analysis was performed in duplicate, and values were averaged. Establishing an α-Cell Line Lacking Catalytically Active PC2—To obtain a neuroendocrine cell line devoid of active PC2 and to better define the contribution of PC2 in the processing of proglucagon in the pancreatic islets, an α-cell line lacking PC2 activity was produced. The Glu2-TAg6 transgenic animal is known to produce pancreatic glucagonomas at a high frequency through expression of the SV40 large T antigen from the rat glucagon promoter (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar, 39Efrat S. Teitelman G. Anwar M. Ruggiero D. Hanahan D. Neuron. 1988; 1: 605-613Google Scholar). Mating Glu2-TAg6 animals to the previously described PC2 null animals (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar) yielded animals that were PC2 homozygous null and carrying the Glu-TAg transgene (see “Materials and Methods”). A single cell line was isolated from the resulting pancreatic α-cell tumors that was characterized further. This cell line has been designated αTC-ΔPC2. Under light microscopic analysis, αTC-ΔPC2 cells exhibit morphological features similar to those of the well characterized αTC1–6 cell line (see Fig. 1, A and B). Furthermore, immunoelectron microscopic analysis demonstrates that, like αTC1–6 cells, αTC-ΔPC2 cells contain numerous secretory granules with (pro)glucagon-like positive immunoreactivity (see Fig. 1, C and D). αTC-ΔPC2 Cells Express a Set of Genes Similar to αTC1–6 Cells and Unlike βTC-3 Cells—To further characterize the new cell line, the expression of several pancreatic islet cell-specific transcripts were defined by RT-PCR. Like αTC1–6 cells, αTC-ΔPC2 cells express proglucagon at high levels, confirming their similarity to normal α-cells (Fig. 2). Like αTC1–6 and unlike the β-cell line βTC-3, αTC-ΔPC2 shows no expression of proinsulin 1 or proinsulin 2. Because the PC2 null allele produces a transcript but no catalytically active PC2 (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar) and the RT-PCR primers used in our analysis exclude the exon 3 region of PC2, all three cell lines produce a PC2 transcript that is of the same size. Also, we have not seen detectable levels of prosomatostatin in either αTC1–6 or αTC-ΔPC2 cells (data not shown). Therefore, like the well characterized αTC1–6 α-cell line, the new αTC-ΔPC2 cells express transcripts known to be found in α-cells (proglucagon and PC2) and fail to express transcripts known not to be expressed in α-cells (proinsulin 1 and 2 and prosomatostatin), suggesting that the new cell line has maintained its α-cell phenotype through cell line derivation. To confirm that the PC2 transcript found in the αTC-ΔPC2 cells corresponds to the null allele, Northern blotting analysis was undertaken. The transcript from the null allele is 114 bp shorter than the wild-type transcript. Blotting for PC2 in βTC3, αTC1–6, and αTC-ΔPC2 cells demonstrates that, as expected and seen in Fig. 2, all three cells express PC2. Furthermore, the transcript for the PC2 null allele demonstrates a slightly increased mobility consistent with its shorter transcript size (Fig. 3A). Anterior pituitary derived AtT20 cells and nonendocrine COS-7 cells, used as controls, do not express detectable levels of PC2. Also, consistent with data from Fig. 2, both α-cell lines express high levels of proglucagon, unlike βTC-3 cells. αTC-ΔPC2 Cells Lack Enzymatically Active PC2—Previous characterizations of islets from PC2 homozygous null mice have demonstrated that the null allele leads to the production of a truncated, inactive proPC2 protein of ∼72 kDa (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar). This protein is not secreted and therefore must be degraded intracellularly (32Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Google Scholar). To confirm the production of this inactive PC2 peptide, immunoblotting was carried out on cell extracts from βTC-3, αTC1–6, and αTC-ΔPC2 cells. Both βTC-3 and αTC1–6 cells show high levels of mature PC2 and smaller levels of proPC2 (75 kDa) as seen previously (28Rouille Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Google Scholar) (see Fig. 3B). The αTC-ΔPC2 cells, however, produce a faster migrating, truncated proPC2 protein, along with lesser amounts of degraded or incorrectly processed peptides, none of which correspond to wild-type active PC2. Interestingly, blots of BiP levels show no significant differences between the PC2 null line and several other neuroendocrine cell lines, indicative of a lack of endoplasmic reticulum stress (see Fig. 3C). Biochemical analysis of culture media from αTC1–6, αTC-ΔPC2, and βTC-3 cell lines for PC2 enzyme activity demonstrates high levels of activity in both αTC1–6 and βTC-3 (Fig. 4). As expected, this activity is inhibited by the PC2-specific inhibitor, 7B2 CT peptide (36Cameron A. Apletalina E.V. Lindberg I. Dalbey R.E. Sigman D.S. Third Ed. The Enzymes. XXII. Academic Press, New York2002: 291-332Google Scholar, 40Lindberg I. van den Hurk W.H. Bui C. Batie C.J. Biochemistry. 1995; 34: 5486-5493Google Scholar, 41Fortenberry Y. Liu J. Lindberg I. J. Neurochem. 1999; 73: 994-1003Google Scholar). On the other hand, αTC-ΔPC2 culture media demonstrated no specific PC2 activity, similar to negative control media from NIH-3T3 cells. These results indicate that the αTC-ΔPC2 cell line maintains an α-cell phenotype in the absence of active PC2 and therefore provides an appropriate model of α-cell function for the study of propeptide processing in the absence of PC2 activity. Proglucagon Processing in αTC-ΔPC2 Cells—The endocrine cell-specific processing of proglucagon is a highly regulated and complex reaction leading to the production of differing mixtures of bioactive peptide hormones, depending on cell type and complement of expressed convertases (28Rouille Y. Westermark G. Martin S.K. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3242-3246Google Scholar, 29Rouille Y. Martin S. Steiner D.F. J. Biol. Chem. 1995; 270: 26488-26496Google Scholar, 30Rouillé Y. Bianchi M. Irminger J.-C. Halban P. FEBS Lett. 1997; 413: 119-123Google Scholar, 31Rouillé Y. Kantengwa S. Irminger J.-C. Halban P.A. J. Biol. Chem. 1997; 272: 32810-32816Google Scholar). Analysis of cellular protein extracts by immunoblotting for proglucagon and glucagon-containing peptide products confirm that αTC1–6 cells efficiently process proglucagon to mature glucagon along with considerable amounts of glicentin and glicentin-related polypeptide-glucagon intermediates (see Fig. 5A). In contrast, αTC-ΔPC2 cells produce no mature glucagon, but instead accumulate large amounts of glicentin. This differs significantly from the nearly complete block in proglucagon processing seen in pancreatic islets of PC2 null animals (42Furuta M. Zhou A. Webb G. Carroll R. Ravazzola M. Orci L. Steiner D.F. J. Biol. Chem. 2001; 276: 27197-27202Google Scholar), suggesting the presence in αTC-ΔPC2 cells of another PC not normally expressed. Pulse-chase analysis of secreted proglucagon-derived peptides confirm that glicentin is a major secreted product along with significant amounts of proglucagon in αTC-ΔPC2 cells at both 45 min and 3 h post-labeling (see Fig. 5B). Significantly, mature glucagon is again not detected. Unlike cellular extracts however, the secreted protein contains a small but significant amount of oxyntomodulin from the αTC-ΔPC2 cells, indicating late or very inefficient processing at the dibasic site Lys31-Arg32. Therefore, the partial processing of the interdomain cleavage site and Lys31-Arg32 led to production of glicentin and oxyntomodulin, but not mature glucagon, from the N-terminal domain of the proglucagon in αTC-ΔPC2 cells. To investigate potential processing of the C-terminal domain of the proglucagon in the αTC-ΔPC2 cells, GLP-1 production was assessed. Using antisera to GLP-1, we demonstrate the presence of both GLP-1-(1–37) and truncated GLP-1 (both 7–37 and 7–36 NH2) at low levels in media from αTC-ΔPC2 cells (see Fig. 5C). In the same cell extracts we detect GLP-1-(1–37) but no truncated forms (see Fig. 5C), indicating late processing at the monobasic site, Arg77. Also, we show the generation of N-terminally extended GLP-2-(111–158) at low levels in αTC-ΔPC2 cells using anti GLP-2 antibody (see Fig. 5D). αTC1–6 cells by contrast secrete mature glucagon with significant amounts of glicentin and glicentin-related polypeptide-glucagon and small amounts of oxyntomodulin (see Fig. 5B), as well as GLP-1 and extended GLP-2, but no truncated GLP-1 fragments (see Fig. 5, C and D). The above results thus demonstrate that efficient early cleavage at the interdomain KR motif (residues 70 and 71) to produce glicentin and later minor cleavages at the dibasic Lys31-Arg32 and Arg109-Arg110 and monobasic Arg77 to generate oxyntomodulin, and the N-terminally extended forms of GLP-1 and GLP-2 and tGLP-1" @default.
• W2000917954 created "2016-06-24" @default.
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• W2000917954 date "2004-07-01" @default.
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• W2000917954 title "Altered Proglucagon Processing in an α-Cell Line Derived from Prohormone Convertase 2 Null Mouse Islets" @default.
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• W2000917954 doi "https://doi.org/10.1074/jbc.m404110200" @default.
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