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Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin
Regulatory Peptides 83 (1999) 123–127
Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin Vidyavathi Udupi Ph.D.a , Heung-Man Lee Ph.D.a , Alexander Kurosky Ph.D.b , b, George H. Greeley Jr. Ph.D. * a
b
Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555 -0725, USA Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555 -0725, USA Received 8 January 1999; received in revised form 23 June 1999; accepted 23 June 1999
Abstract The purpose of this study was to test the hypothesis that the endoprotease, prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST). A human pancreatic carcinoid cell line (BON) that expresses PC-1, CGA and PST was stably transfected with antisense PC-1 mRNA. BON cells expressing antisense PC-1 mRNA showed nearly complete abolishment of PC-1 protein ( | 95% reduction) and an 80% reduction in cell content of PST immunoreactivity (PST-IR) as assessed by high-performance liquid chromatography in combination with measurement of PST-IR. These findings indicate that PC-1 is essential for processing CGA to PST. 1999 Elsevier Science B.V. All rights reserved. Keywords: Peptide; Processing; Endoprotease; Antisense
1. Introduction Chromogranin A (CGA) is an acidic secretory protein produced in nearly all endocrine and neuroendocrine cells [1–3]. CGA is thought to be a precursor protein (i.e. prohormone) since it contains multiple, conserved basic amino acid pairs [4–7]. Such pairs of basic amino acids can serve as cleavage sites to give smaller, biologically active peptides. Additional evidence supporting the role of CGA as a precursor protein was given by the isolation of a peptide from porcine duodenal extracts called pancreastatin (PST) [8]. PST is homologous to a carboxy-region of CGA. The PST sequence in human CGA occurs in residues 250–301 and is flanked by either single or paired basic amino acid residues at the amino and carboxy ends [9] (see Fig. 1). PST is amidated at the carboxy terminal. *Corresponding author. Tel.: 1 1-409-772-2094; fax: 1 1-409-7726368. E-mail address: [email protected] (G.H. Greeley Jr.)
PST displays a variety of biological activities in the gastrointestinal tract, pancreas and in other tissues. PST can inhibit pancreatic insulin and exocrine secretion [8,10,11], and PST can stimulate gastric acid secretion in dogs [12]. CGA and parathyroid hormone (PTH) secretion are inhibited by PST in cultured parathyroid cells [13]. Proforms of peptide hormones and neuropeptides are processed by a selective cleavage at the C-terminal side of paired basic amino acids by specific endoproteases called prohormone convertases [14,15]. To date, seven members of the endoprotease family have been identified, including prohormone convertase-1 (PC-1) (also called PC-3), PC-2, -4, -5 (also called PC-6), PC-7, PACE-4 and furin [14–17]. PC-1, PC-2 and PC-5 are involved primarily in processing endocrine and neuroendocrine prohormones [14,15,17,18]. We have characterized a novel human pancreatic carcinoid cell line, called BON, that synthesizes and releases CGA, PST and neurotensin [19,20]. BON cells express furin and PC-1, but not PC-2 or PC-5 / 6 ([21], see Fig. 2). BON cells, therefore, represent a model to examine the
0167-0115 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00061-0
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Fig. 1. Schematic of human chromogranin A (CGA) showing localization of pancreastatin (PST) and single and paired basic amino acids in the vicinity of PST.
Fig. 2. Northern blotting analysis of BON cell RNA showing expression of furin. PC-2 and PC-5 / 6 are not expressed in BON cells (not shown). A 10-mg amount of poly [A1 ] RNA was hybridized with a rat 32 P-labeled furin cRNA probe. mRNA was visualized by autoradiography.
relevance of PC-1 in the formation of PST from CGA. The purpose of this study was to examine the role of PC-1 in the processing of CGA to PST by stable expression of antisense PC-1 mRNA.
2. Materials and methods
2.1. Materials Trypsin and lipofectamine were obtained from GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone Labs. (Logan, UT, USA). The antisense PC-1 expression plasmid was a gift from M. Beinfeld (Tufts University Medical School, Boston, MA, USA). Human PST-52 (1–52) and human PST-29 (24–52) were purchased from Peninsula Labs. (San Carlos, CA, USA).
structed by inserting the first 491 bases of the PC-1 cDNA into the mammalian expression vector pCMV5 in the antisense orientation. Expression of antisense PC-1 mRNA is driven constitutively by the cytomegalovirus promoter. pCMV/ anti-PC-1 was cotransfected with pMtNeo (3:1 molar ratio), which confers resistance to the antibiotic G418. BON cells transfected with pCMV5 / antiPC-1 were selected with G-418. Control BON cell lines were produced by transfection with pMtNeo and pCMV5 plasmid, without antisense PC-1 mRNA insert, and selected with G-418.
2.2.2. Western blotting analysis of PC-1 protein levels Western analysis was performed as described earlier [22]. Briefly, 50 mg BON cell protein extracts [extracted in RIPA-lysis buffer consisting of phosphate-buffered saline (PBS) (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride, pH 7.4) 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 100 units / ml aprotinin] were subjected to electrophoresis on 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel. Proteins were transferred to a membrane, blocked with 5% nonfat milk in Trisbuffered saline (TBS) (50 mM Tris, 200 mM NaCl, pH 7.4). Membranes were then incubated with PC-1 antiserum (1:1000 dilution) followed by a second incubation in HRPconjugated antirabbit secondary antibody (1:3000 dilution) and developed using the ECL method (Amersham). A polyclonal antiserum that recognizes PC-1 was a gift from Dr. I. Lindberg (Louisiana State Medical Center, New Orleans, LA, USA).
2.2. Methods 2.2.1. Maintenance and transfection of BON cells BON cells were routinely cultured in DMEM and F12K (1:1, Mediatech, Herndon, VA, USA) medium containing 5% FBS. BON cells were transfected with antisense PC-1 expression plasmid using the lipofectamine method. The antisense PC-1 plasmid pCMV5 / anti-PC-1 was con-
2.2.3. High-performance liquid chromatographic separation of pancreastatin immunoreactivity Cells from four to eight 10-cm plates were pooled and extracted by sonicating in 1 M acetic acid containing 5 mM ethylenediaminetetraacetic acid (EDTA), aprotinin (100 units / ml) and PMSF (1 mM). After lyophilizing cell supernatants to dryness, cell extracts were reconstituted in
V. Udupi et al. / Regulatory Peptides 83 (1999) 123 – 127
4 M guanidine chloride and applied to reversed-phase high-performance liquid chromatography (HPLC) (Vydac C 18 column, 25 cm 3 2.5 mm, Applied Biosystems, Foster City, CA, USA) equilibrated with 0.1% trifluoroacetic acid in 20% acetonitrile. Peptides were eluted with a linear gradient of 20–60% acetonitrile over 90 min at 200 ml / min. Acetonitrile was removed by evaporation using a speedvac. HPLC fractions were then assayed for immunoreactive PST (PST-IR) contents by a PST radioimmunoassay (RIA) using a procedure described in detail previously [19]. This PST RIA uses a PST antiserum that was generated against synthetic porcine PST (1–49). It crossreacts with human PST (1–52) and PST (24–52), but not with CGA.
3. Results Transfection of BON cells with the antisense PC-1
Fig. 3. Reduction of prohormone convertase-1 (PC-1) protein expression in BON cells that express antisense PC-1 mRNA. Fifty-mg of cellular protein from normal BON cells, BON cells stably transfected with antisense PC-1 mRNA (14M) or BON cells transfected with the empty vector (MC-9) were immunoblotted with a polyclonal PC-1 antiserum (MW 5 molecular mass).
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mRNA expression plasmid resulted in generation of multiple BON clones which survived G-418 selection and expressed decreased levels of PC-1 protein (data not shown). None of the transfected BON cells showed any gross morphological changes and grew at rates similar to those of normal BON cells. A single clone, called 14M, was used in this study. In normal BON cells, PC-1 occurs in two forms, molecular mass (Mr ) | 66 000 and | 84 000 (Fig. 3). In control BON cells (called MC-9) transfected with the empty pCMV5 vector, the variants and magnitude of PC-1 protein expression were unchanged when compared to control BON cells. Constitutive expression of antisense PC-1 mRNA in 14M BON cells resulted in a near complete abolishment of PC-1 protein expression. In BON cells expressing antisense PC-1 mRNA, PC-1 protein levels are reduced by | 95% when compared to PC-1 protein levels of normal BON cells or to BON cells expressing the pCMV5 vector only (i.e. with no antisense PC-1 mRNA insert). In BON cells expressing antisense PC-1, the larger Mr | 84 000 variant was absent whereas the Mr | 66 000 form is present in reduced amounts. Examination of PST-IR levels by PST RIA in BON cells expressing the pCMV5 vector alone (MC-9) showed that PST-IR eluted primarily in two major immunoreactive peaks on HPLC (Fig. 4). PST-IR contained in BON cell extracts eluted at fractions | 17 and | 20. The first peak (i.e., fraction | 20) coeluted with the PST-52 standard whereas the second peak (i.e., fraction | 17) eluted between the PST-52 and PST (24–52) standards. In BON cells expressing antisense PC-1 mRNA (14M BON cells), production of PST-IR was nearly abolished; a greatly reduced PST-IR peak was detected at fraction 17, and the larger peak ( | fraction 20) observed in control BON cells was completely absent.
Fig. 4. Effect of depressed PC-1 protein expression on PST-IR in BON cells. In BON cells expressing the antisense PC-1 mRNA (14 M), PST-IR is reduced by | 80%. PST-IR was extracted from BON cells by sonicating cells in 1 M acetic acid containing 5 mM EDTA, aprotinin (100 units / ml) and 1 mM PMSF. PST-IR contained in extracts of BON cells was separated by HPLC and measured by a PST radioimmunoassay. The broken line (- - -) running between the left and right axes indicates the acetonitrile gradient. The elution positions of hPST (1–52) and hPST (24–52) are indicated.
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4. Discussion
Acknowledgements
The primary finding of this paper is that the prohormone convertase-1 (PC-1) is essential for processing of CGA to PST. This was demonstrated clearly by a substantial reduction of PST-IR in BON cells ( | 80%) having a reduced expression of PC-1 protein levels ( | 95% reduction). Antisense message expression has been used previously to inhibit prohormone convertase expression by Yoon and Beinfeld [23] to show the role of PC-2 in the processing of pro-cholecystokinin (CCK) to CCK-22, and by Bloomquist et al. [24] to show a role for PC-1 in processing of POMC in AtT-20 cells. It is noteworthy, in these earlier studies as well as in ours, that complete abolishment of cellular PC protein expression is not necessary to demonstrate their specific involvement in processing of prohormones to smaller peptides. Interestingly, in our experiments, antisense PC-1 mRNA expression reduced the amount of Mr | 66 000 PC-1 form, but the Mr | 84 000 form was completely absent. This finding suggests that exceptionally marginal amounts of the Mr | 84 000 form are produced which are evidently processed immediately to the Mr | 66 000 form. BON cells express furin, and PC-1 ([22], unreported findings). In the present study, our findings indicate that furin did not assume the role of PC-1 for processing CGA to PST in BON cells expressing antisense PC-1 mRNA. A chronic reduction of PC-1 protein expression might result in replacement of PC-1 function by furin since furin can be involved in processing of endocrine and neuroendocrine precursor proteins to biologically-active peptides [25]. The biosynthetic relationship between PST and CGA is supported by the finding that the PST sequence in human CGA is flanked by either single or paired basic amino acids (i.e., Arg, Lys,) at the amino and carboxyl terminals. In human CGA, the PST-52 sequence is flanked by Arg 248 and Lys 249 at the amino end and by Lys 303 at the carboxyl end. These are potential cleavage sites for a prohormone convertase, presumably PC-1, based on the present findings. Endoproteolytic cleavage by PCs at single basic amino acid residues can also occur [26]. Our study as well as others have found variants of PST-IR, including PST-29 (273–301), PST-52 (250–301), PST-92 (210–301), and PST-186 (116–301) [27–30], suggesting that CGA is cleaved at several single basic or paired basic amino acids to form PST variants or PST biosynthetic intermediates. There are single basic amino acids at positions 175 (Lys), 195 (Arg) and 197 (Lys) and two additional pairs of basic residues at positions 322, 323 (Lys, Arg) and 338, 339 (Lys, Arg) in human CGA. Earlier studies also indicate that PST processing from CGA occurs with intermediate forms [31,32]. For instance, in enteroendocrine cells of the rat small intestine and stomach fundus, processing to PST is incomplete, with the primary variant occurring as a C-terminally extended form, whereas in enteroendocrine cells of the stomach antrum and pancreatic islets, Cterminally amidated PST occurs primarily.
Supported by a grant from the National Institutes of Health (PO1-DK35608).
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[26] Nakayama K, Watanabe T, Nakagawa T, Kim W-S, Nagahama M, Hosaka M, Hatsuzawa K, Kondoh-Hashiba K, Murakami K. Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites. J Biol Chem 1992;267:16335–40. [27] Sekiya K, Ghatei MA, Minamino N, Bretherton-Watt D, Matsuo H, Bloom SR. Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma. FEBS Lett 1988;228:153–6. [28] Sekiya K, Ghatei MA, Minamino N, Bretherton-Watt D, Matsuo H, Bloom SR. Isolation of human pancreastatin fragment containing the active sequence from a glucagonoma [published erratum]. FEBS Lett 1988;231:451. [29] Schmidt WE, Siegel EG, Kratzin H, Creutzfeldt W. Isolation and primary structure of tumor-derived peptides related to human pancreastatin and chromogranin A. Proc Natl Acad Sci USA 1988;85:8231–5. [30] Tamamura H, Ohta M, Yoshizawa K, Ono Y, Funakoshi A, Miyasaka K, Tateishi K, Jimi A, Yajima H, Fujii N, Funakoshi S. Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48. Eur J Biochem 1990;191:33–9. [31] Arden SD, Rutherford NG, Guest PC, Curry WJ, Bailyes EM, Johnston CF, Hutton JC. The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2. Biochem J 1994;298:521–8. [32] Watkinson A, Jonsson A-C, Davison M, Young J, Lee CM, Moore S, Dockray GJ. Heterogeneity of chromogranin A-derived peptides in bovine gut, pancreas and adrenal medulla. Biochem J 1991;276:471–9.
Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin Vidyavathi Udupi Ph.D.a , Heung-Man Lee Ph.D.a , Alexander Kurosky Ph.D.b , b, George H. Greeley Jr. Ph.D. * a
b
Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555 -0725, USA Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77555 -0725, USA Received 8 January 1999; received in revised form 23 June 1999; accepted 23 June 1999
Abstract The purpose of this study was to test the hypothesis that the endoprotease, prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST). A human pancreatic carcinoid cell line (BON) that expresses PC-1, CGA and PST was stably transfected with antisense PC-1 mRNA. BON cells expressing antisense PC-1 mRNA showed nearly complete abolishment of PC-1 protein ( | 95% reduction) and an 80% reduction in cell content of PST immunoreactivity (PST-IR) as assessed by high-performance liquid chromatography in combination with measurement of PST-IR. These findings indicate that PC-1 is essential for processing CGA to PST. 1999 Elsevier Science B.V. All rights reserved. Keywords: Peptide; Processing; Endoprotease; Antisense
1. Introduction Chromogranin A (CGA) is an acidic secretory protein produced in nearly all endocrine and neuroendocrine cells [1–3]. CGA is thought to be a precursor protein (i.e. prohormone) since it contains multiple, conserved basic amino acid pairs [4–7]. Such pairs of basic amino acids can serve as cleavage sites to give smaller, biologically active peptides. Additional evidence supporting the role of CGA as a precursor protein was given by the isolation of a peptide from porcine duodenal extracts called pancreastatin (PST) [8]. PST is homologous to a carboxy-region of CGA. The PST sequence in human CGA occurs in residues 250–301 and is flanked by either single or paired basic amino acid residues at the amino and carboxy ends [9] (see Fig. 1). PST is amidated at the carboxy terminal. *Corresponding author. Tel.: 1 1-409-772-2094; fax: 1 1-409-7726368. E-mail address: [email protected] (G.H. Greeley Jr.)
PST displays a variety of biological activities in the gastrointestinal tract, pancreas and in other tissues. PST can inhibit pancreatic insulin and exocrine secretion [8,10,11], and PST can stimulate gastric acid secretion in dogs [12]. CGA and parathyroid hormone (PTH) secretion are inhibited by PST in cultured parathyroid cells [13]. Proforms of peptide hormones and neuropeptides are processed by a selective cleavage at the C-terminal side of paired basic amino acids by specific endoproteases called prohormone convertases [14,15]. To date, seven members of the endoprotease family have been identified, including prohormone convertase-1 (PC-1) (also called PC-3), PC-2, -4, -5 (also called PC-6), PC-7, PACE-4 and furin [14–17]. PC-1, PC-2 and PC-5 are involved primarily in processing endocrine and neuroendocrine prohormones [14,15,17,18]. We have characterized a novel human pancreatic carcinoid cell line, called BON, that synthesizes and releases CGA, PST and neurotensin [19,20]. BON cells express furin and PC-1, but not PC-2 or PC-5 / 6 ([21], see Fig. 2). BON cells, therefore, represent a model to examine the
0167-0115 / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0167-0115( 99 )00061-0
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Fig. 1. Schematic of human chromogranin A (CGA) showing localization of pancreastatin (PST) and single and paired basic amino acids in the vicinity of PST.
Fig. 2. Northern blotting analysis of BON cell RNA showing expression of furin. PC-2 and PC-5 / 6 are not expressed in BON cells (not shown). A 10-mg amount of poly [A1 ] RNA was hybridized with a rat 32 P-labeled furin cRNA probe. mRNA was visualized by autoradiography.
relevance of PC-1 in the formation of PST from CGA. The purpose of this study was to examine the role of PC-1 in the processing of CGA to PST by stable expression of antisense PC-1 mRNA.
2. Materials and methods
2.1. Materials Trypsin and lipofectamine were obtained from GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone Labs. (Logan, UT, USA). The antisense PC-1 expression plasmid was a gift from M. Beinfeld (Tufts University Medical School, Boston, MA, USA). Human PST-52 (1–52) and human PST-29 (24–52) were purchased from Peninsula Labs. (San Carlos, CA, USA).
structed by inserting the first 491 bases of the PC-1 cDNA into the mammalian expression vector pCMV5 in the antisense orientation. Expression of antisense PC-1 mRNA is driven constitutively by the cytomegalovirus promoter. pCMV/ anti-PC-1 was cotransfected with pMtNeo (3:1 molar ratio), which confers resistance to the antibiotic G418. BON cells transfected with pCMV5 / antiPC-1 were selected with G-418. Control BON cell lines were produced by transfection with pMtNeo and pCMV5 plasmid, without antisense PC-1 mRNA insert, and selected with G-418.
2.2.2. Western blotting analysis of PC-1 protein levels Western analysis was performed as described earlier [22]. Briefly, 50 mg BON cell protein extracts [extracted in RIPA-lysis buffer consisting of phosphate-buffered saline (PBS) (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride, pH 7.4) 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 100 units / ml aprotinin] were subjected to electrophoresis on 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel. Proteins were transferred to a membrane, blocked with 5% nonfat milk in Trisbuffered saline (TBS) (50 mM Tris, 200 mM NaCl, pH 7.4). Membranes were then incubated with PC-1 antiserum (1:1000 dilution) followed by a second incubation in HRPconjugated antirabbit secondary antibody (1:3000 dilution) and developed using the ECL method (Amersham). A polyclonal antiserum that recognizes PC-1 was a gift from Dr. I. Lindberg (Louisiana State Medical Center, New Orleans, LA, USA).
2.2. Methods 2.2.1. Maintenance and transfection of BON cells BON cells were routinely cultured in DMEM and F12K (1:1, Mediatech, Herndon, VA, USA) medium containing 5% FBS. BON cells were transfected with antisense PC-1 expression plasmid using the lipofectamine method. The antisense PC-1 plasmid pCMV5 / anti-PC-1 was con-
2.2.3. High-performance liquid chromatographic separation of pancreastatin immunoreactivity Cells from four to eight 10-cm plates were pooled and extracted by sonicating in 1 M acetic acid containing 5 mM ethylenediaminetetraacetic acid (EDTA), aprotinin (100 units / ml) and PMSF (1 mM). After lyophilizing cell supernatants to dryness, cell extracts were reconstituted in
V. Udupi et al. / Regulatory Peptides 83 (1999) 123 – 127
4 M guanidine chloride and applied to reversed-phase high-performance liquid chromatography (HPLC) (Vydac C 18 column, 25 cm 3 2.5 mm, Applied Biosystems, Foster City, CA, USA) equilibrated with 0.1% trifluoroacetic acid in 20% acetonitrile. Peptides were eluted with a linear gradient of 20–60% acetonitrile over 90 min at 200 ml / min. Acetonitrile was removed by evaporation using a speedvac. HPLC fractions were then assayed for immunoreactive PST (PST-IR) contents by a PST radioimmunoassay (RIA) using a procedure described in detail previously [19]. This PST RIA uses a PST antiserum that was generated against synthetic porcine PST (1–49). It crossreacts with human PST (1–52) and PST (24–52), but not with CGA.
3. Results Transfection of BON cells with the antisense PC-1
Fig. 3. Reduction of prohormone convertase-1 (PC-1) protein expression in BON cells that express antisense PC-1 mRNA. Fifty-mg of cellular protein from normal BON cells, BON cells stably transfected with antisense PC-1 mRNA (14M) or BON cells transfected with the empty vector (MC-9) were immunoblotted with a polyclonal PC-1 antiserum (MW 5 molecular mass).
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mRNA expression plasmid resulted in generation of multiple BON clones which survived G-418 selection and expressed decreased levels of PC-1 protein (data not shown). None of the transfected BON cells showed any gross morphological changes and grew at rates similar to those of normal BON cells. A single clone, called 14M, was used in this study. In normal BON cells, PC-1 occurs in two forms, molecular mass (Mr ) | 66 000 and | 84 000 (Fig. 3). In control BON cells (called MC-9) transfected with the empty pCMV5 vector, the variants and magnitude of PC-1 protein expression were unchanged when compared to control BON cells. Constitutive expression of antisense PC-1 mRNA in 14M BON cells resulted in a near complete abolishment of PC-1 protein expression. In BON cells expressing antisense PC-1 mRNA, PC-1 protein levels are reduced by | 95% when compared to PC-1 protein levels of normal BON cells or to BON cells expressing the pCMV5 vector only (i.e. with no antisense PC-1 mRNA insert). In BON cells expressing antisense PC-1, the larger Mr | 84 000 variant was absent whereas the Mr | 66 000 form is present in reduced amounts. Examination of PST-IR levels by PST RIA in BON cells expressing the pCMV5 vector alone (MC-9) showed that PST-IR eluted primarily in two major immunoreactive peaks on HPLC (Fig. 4). PST-IR contained in BON cell extracts eluted at fractions | 17 and | 20. The first peak (i.e., fraction | 20) coeluted with the PST-52 standard whereas the second peak (i.e., fraction | 17) eluted between the PST-52 and PST (24–52) standards. In BON cells expressing antisense PC-1 mRNA (14M BON cells), production of PST-IR was nearly abolished; a greatly reduced PST-IR peak was detected at fraction 17, and the larger peak ( | fraction 20) observed in control BON cells was completely absent.
Fig. 4. Effect of depressed PC-1 protein expression on PST-IR in BON cells. In BON cells expressing the antisense PC-1 mRNA (14 M), PST-IR is reduced by | 80%. PST-IR was extracted from BON cells by sonicating cells in 1 M acetic acid containing 5 mM EDTA, aprotinin (100 units / ml) and 1 mM PMSF. PST-IR contained in extracts of BON cells was separated by HPLC and measured by a PST radioimmunoassay. The broken line (- - -) running between the left and right axes indicates the acetonitrile gradient. The elution positions of hPST (1–52) and hPST (24–52) are indicated.
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4. Discussion
Acknowledgements
The primary finding of this paper is that the prohormone convertase-1 (PC-1) is essential for processing of CGA to PST. This was demonstrated clearly by a substantial reduction of PST-IR in BON cells ( | 80%) having a reduced expression of PC-1 protein levels ( | 95% reduction). Antisense message expression has been used previously to inhibit prohormone convertase expression by Yoon and Beinfeld [23] to show the role of PC-2 in the processing of pro-cholecystokinin (CCK) to CCK-22, and by Bloomquist et al. [24] to show a role for PC-1 in processing of POMC in AtT-20 cells. It is noteworthy, in these earlier studies as well as in ours, that complete abolishment of cellular PC protein expression is not necessary to demonstrate their specific involvement in processing of prohormones to smaller peptides. Interestingly, in our experiments, antisense PC-1 mRNA expression reduced the amount of Mr | 66 000 PC-1 form, but the Mr | 84 000 form was completely absent. This finding suggests that exceptionally marginal amounts of the Mr | 84 000 form are produced which are evidently processed immediately to the Mr | 66 000 form. BON cells express furin, and PC-1 ([22], unreported findings). In the present study, our findings indicate that furin did not assume the role of PC-1 for processing CGA to PST in BON cells expressing antisense PC-1 mRNA. A chronic reduction of PC-1 protein expression might result in replacement of PC-1 function by furin since furin can be involved in processing of endocrine and neuroendocrine precursor proteins to biologically-active peptides [25]. The biosynthetic relationship between PST and CGA is supported by the finding that the PST sequence in human CGA is flanked by either single or paired basic amino acids (i.e., Arg, Lys,) at the amino and carboxyl terminals. In human CGA, the PST-52 sequence is flanked by Arg 248 and Lys 249 at the amino end and by Lys 303 at the carboxyl end. These are potential cleavage sites for a prohormone convertase, presumably PC-1, based on the present findings. Endoproteolytic cleavage by PCs at single basic amino acid residues can also occur [26]. Our study as well as others have found variants of PST-IR, including PST-29 (273–301), PST-52 (250–301), PST-92 (210–301), and PST-186 (116–301) [27–30], suggesting that CGA is cleaved at several single basic or paired basic amino acids to form PST variants or PST biosynthetic intermediates. There are single basic amino acids at positions 175 (Lys), 195 (Arg) and 197 (Lys) and two additional pairs of basic residues at positions 322, 323 (Lys, Arg) and 338, 339 (Lys, Arg) in human CGA. Earlier studies also indicate that PST processing from CGA occurs with intermediate forms [31,32]. For instance, in enteroendocrine cells of the rat small intestine and stomach fundus, processing to PST is incomplete, with the primary variant occurring as a C-terminally extended form, whereas in enteroendocrine cells of the stomach antrum and pancreatic islets, Cterminally amidated PST occurs primarily.
Supported by a grant from the National Institutes of Health (PO1-DK35608).
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