Proinsulin Processing by the Subtilisin-Related Proprotein Convertases Furin, PC2, and PC3 (Prohormone/Kex2 Protease/Vaccinia Vectors/Islets of Langerhans) STEVEN P
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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 8822-8826, September 1992 Biochemistry Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3 (prohormone/Kex2 protease/vaccinia vectors/islets of Langerhans) STEVEN P. SMEEKENS*t*, ANTHONY G. MONTAG§, GARY THOMAS1, CORRINE ALBIGES-RIzO*II, RAYMOND CARROLL*, MICHELLE BENIGt, LISA A. PHILLIPS*, SEAN MARTIN*, SHINYA OHAGIt, PAUL GARDNER*, HEWSON H. SWIFT**, AND DONALD F. STEINER*t *The Howard Hughes Medical Institute, The Departments of tBiochemistry and Molecular Biology, and §Pathology and the **Erman Cell Biological Laboratories, The University of Chicago, Chicago, IL 60637; and MThe Vollum Institute, Portland, OR 97201 Contributed by Donald F. Steiner, June 19, 1992 ABSTRACT Experiments using recombinant vaccinia vi- specifically in pancreatic islets, pituitary, and other neuro- ruses expressing rat proinsulin I coinfected into COS-7 cells endocrine cells (12-16) and have been shown by heterologous with recombinant vaccinia virus expressing human furin, gene transfer experiments to be capable of cleaving selec- human PC2, mouse PC3 (subtilisin-related proprotein conver- tively at the Lys-Arg and Arg-Arg processing sites within tases 1-3, respectively), or yeast Kex2 indicate that in this precursors such as proopiomelanocortin and prorenin (17- system both Kex2 and furin produce mature insulin, whereas 20). PC2 selectively cleaves proinsulin at the C-peptide-A-chain A third member of this superfamily, furin** (also called junction. This is a property consistent with its probable identity PACE), has also been shown to correctly process a variety of with the rat insulinoma granule type II proinsulin processing precursors, including the 3-nerve growth factor and von activity as described by Davidson et aL [Davidson, H. W., Willebrand factor precursors, both of which have processing Rhodes, C. J. & Hutton, J. C. (1988) Nature (London) 333, recognition sequences consisting minimally ofArg-Xaa-Xaa- 93-96]. PC3 generates mature insulin but cleaves preferen- Arg (4, 5, 21-23). However, in contrast to PC2 and PC3, tially at the proinsulin B-chain-C-peptide junction. This pat- furin/PACE is expressed in many tissues of the body and tern of cleavage by PC3 is similar, but not identical, to that of appears to be predominantly localized to the Golgi region in the highly B-chain-C-peptide junction-selective type I activity cells (4, 23). Like Kex2, furin has a membrane-spanning as described by Davidson et aL, perhaps due to the presence of sequence near the C terminus that is necessary for its Golgi a P4 arginine residue near the C-peptide-A-chain junction retention (6, 24, 25). Since many of its substrates are moved unique to the rat proinsulins. These results along with data to the cell surface or secreted by small constitutive (or presented on the expression ofboth PC2 and PC3 in islet 13 cells unregulated) vesicles, furin-or enzymes like it, such as the strongly support the conclusion that these proteases are in- recently identified PACE4 (26)-appears to account for the volved in the conversion of proinsulin to insulin in vivo. widespread ability of cells to process growth factor and receptor precursor molecules while lacking the ability to Highly specific proteolysis at sites marked by basic amino efficiently process most prohormones. acids is an important requirement for the maturation ofa large In the experiments reported here, we demonstrate that PC2 number of protein precursors (1, 2). Proinsulin was the first and PC3 are localized in the pancreas to the islets of Langer- of these precursors to be identified and characterized (3). hans and that both proteases selectively process specific Since that time, >150 proteins and peptides have been found cleavage sites in proinsulin when coexpressed with rat pro- to require such processing in eukaryotic organisms as diverse insulin I by means of vaccinia virus vectors. These results as yeast and humans (2). These proproteins include virtually support the conclusion that these proteases are involved in all neuropeptides and peptide hormones, as well as a large the conversion of proinsulin to insulin in the maturing secre- and diverse family of membrane and viral glycoproteins, tory granules and lend further support to our claim that PC2 growth factors, and plasma proteins (4-6). and PC3 represent a core of endoproteases responsible for Despite intensive efforts, it is only recently that several prohormone maturation at paired basic residue sites in a endoproteases that carry out some of these cleavages have number of neuroendocrine cells (17). been identified and characterized. Progress in this field was stimulated by the identification in yeast ofthe Kex2 protease, MATERIALS AND METHODS a membrane-bound dibasic amino acid-specific endoprotease required for maturation of the pro-a-factor and killer toxin Antisera. Antisera were raised to peptides corresponding peptides (7, 8). More recently, a superfamily of mammalian to amino acids 65-77 and 108-120 of preproPC2 and amino serine proteases has been identified in animal cells, all acids 95-108 and 110-122 of preproPC3. The two PC2 or the members of which have structural similarity to the Kex2 two PC3 peptides were both conjugated as a mixture to endoprotease (9). Interestingly, both the yeast and mamma- keyhole limpet hemocyanin via added C- or N-terminal lian proteases contain catalytic domains that are organized cysteine residues and were then injected with adjuvants for similarly to those of the bacterial subtilisins, i.e., having the immunization into rabbits, essentially as described (27). Sera characteristic order and spacing ofthe catalytically important collected 10 days after each booster injection were assessed aspartate, histidine, and serine residues of this special class of serine proteases (10, 11). Two of these mammalian en- *Present address: Chiron Corporation, Emeryville, CA 94608. zymes, PC2** and PC3** (also called PC1), are expressed IIPresent address: Laboratoire D-Etude des Systems Adhesifs Cel- lulares, Universite J. Fourier, Grenoble, France. **To avoid confusion due to wide usage ofthe designation "PC," we The publication costs of this article were defrayed in part by page charge have recently proposed that these proteinases be designated payment. This article must therefore be hereby marked "advertisement" substilisin-related proprotein convertases (SPCs): i.e., SPC1, fu- in accordance with 18 U.S.C. §1734 solely to indicate this fact. rin; SPC2, PC2; SPC3, PC3; SPC4, PACE 4 (51). 8822 Downloaded by guest on October 1, 2021 Biochemistry: Smeekens et al. Proc. Natl. Acad. Sci. USA 89 (1992) 8823 for binding activity either to the original peptides immobilized oped over 75 min at a flow rate of1.0 ml/min; 1.0-ml fractions in 96-well titer plates by ELISA (28) or to PC2 or PC3 were collected. Low pressure chromatography was used to preparations derived from a human insulinoma or cultured verify the composition of peptides resolved by HPLC, as AtT20 cells, respectively, by Western blot analysis (29). follows. Mature insulin was separated from proinsulin and Immunoreactivity was detected using the ECL chemilumi- the des-(31-32)- and des-(64-65)-proinsulin processing inter- nescence reaction (Amersham). mediates by P-30 gel chromatography in 3 M acetic acid/ Vaccinia Viruses. The Rat I preproinsulin cDNA (30) was bovine serum albumin (50 pg/ml) as described (33). Cleavage cloned into the vector pVZneo (31) and the resulting pVZ- at one or both processing sites within proinsulin was assessed neo:rInsI construct was used to introduce the Rat I prepro- by the presence of the normal or modified B- or A-chains of insulin sequence into the vaccinia virus genome (strain WR) insulin determined after oxidative sulfitolysis of the samples by homologous recombination as described (32). Recombi- and chromatography on Sephadex G-75 in 50o acetic acid as nant vaccinia viruses encoding human PC2, mouse PC3, described (34). Human insulin, proinsulin, and the des-(31- human furin, and Kex2 have been described (17). 32)- and des-(64-65)-proinsulin intermediates were used as Cell Culture and Expression. COS-7 cells were maintained standards (kindly provided by Bruce Frank and Ronald in Dulbecco's modified Eagle's medium (DMEM) containing Chance, Eli Lilly). 10%o (vol/vol) fetal calf serum and incubated in a humidified incubator at 370C and 5% C02/95% air. For expression studies, COS-7 cells were plated at 1 x 106 cells per 100-mm RESULTS plate and infected with recombinant vaccinia virus at a The original identification and isolation ofPC2 from a human multiplicity of infection of 5 as described (17). After incuba- insulinoma (12) and the finding by Northern blot analysis that tion for 6 hr, medium was removed and replaced with DMEM PC2 and PC3 are expressed in both insulinoma tissue and minus cysteine but supplemented with 100 pACi of [35S]cys- isolated islets ofLangerhans (13) led us to investigate the role teine (1 Ci = 37 GBq), and the cells were incubated further of these enzymes in the proteolytic maturation of proinsulin. for 18 hr. The medium was then collected, any remaining cells Immunohistochemical analysis of rat pancreas sections and debris were removed by centrifugation, and the insulin- showed that both the PC2 and PC3 antisera react specifically related material was immunopurified using guinea pig anti- with the majority of cells within islets of Langerhans, the site insulin antiserum coupled to Affi-Gel 10 agarose beads (Bio- of synthesis and proteolytic processing of a number of Rad). Briefly, 10 ml of labeled medium from each 100-mm peptide hormones including proinsulin, proglucagon, and plate was mixed with 0.2 ml of a 50%o slurry of antiserum- prosomatostatin (Fig. 1 A and B). No immunostaining was coupled beads in phosphate-buffered saline (PBS) in a sealed observed within the exocrine pancreas. The diffuse islet Econo column (Bio-Rad) and gently rocked overnight at 4°C.