Miniglucagon (MG)-Generating Endopeptidase, which Processes Glucagon into MG, Is Composed of N-Arginine Dibasic Convertase and Aminopeptidas
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内分泌学杂志 2005年第2期
Institut National de la Santé et de la Recherche Médicale Unité 376 (G.F., F.B., S.D., D.L.-N., D.B.), Centre Hospitalier Universitaire Arnaud de Villeneuve, and Centre Régional d’Imagerie Cellulaire (F.T.), 34295 Montpellier, Cedex 5, France; Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5160 (A.-D.L., R.G.), Institut de Biologie, 34060 Montpellier, Cedex 1, France; CNRS FRE2621 (S.C., T.F.), Université Pierre et Marie Curie, 75006 Paris, France; and Laboratory of Biochemical Neuroendocrinology (A.P.), Clinical Research Institute of Montreal, Montreal, Canada H2W 1R7
Address all correspondence and requests for reprints to: Dominique Bataille, Institut National de la Santé et de la Recherche Médicale Unité 376, Centre Hospitalier Universitaire Arnaud de Villeneuve, 371, Rue du Doyen G. Giraud, 34295 Montpellier, Cedex 5, France. E-mail: bataille@montp.inserm.fr.
Abstract
Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17–Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing -cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting TC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse TC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the TC1.6 and native rat -cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of -cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
Introduction
A WIDE VARIETY of peptidic hormones and regulatory peptides are produced from larger proteins, called prohormones (1). A typical example of a prohormone is proglucagon, a 160-amino-acid peptide from which originate several hormones with different biological roles (2, 3, 4) through tissue-specific posttranslational processing. In the -cells, the 29-amino-acid peptide glucagon is its major end product (5), but a secondary posttranslational processing occurs that leads to the C-terminal (19–29)-fragment of glucagon, called miniglucagon (MG) (6, 7). This peptide displays original properties opposite to those of glucagon, the mother-hormone; among them figure a negative inotropic action on embryonic chick cardiac cells (8), inhibition of the liver plasma membrane calcium pump (9), and interestingly, an inhibitory effect on secretagogue-induced insulin secretion from ?-cells (7, 10). The posttranslational processing at the Arg17–Arg18 dibasic site of glucagon leading to MG was demonstrated to occur in the glucagon-producing -cells of the islets of Langerhans (10) but also from circulating glucagon in heart and liver (8, 11). In both cases, the small proportion of MG produced is more than enough to be biologically relevant, because of the huge potency of MG compared with glucagon (7). Classically, precursors containing basic residues arranged in doublets, such as Lys-Lys, Arg-Arg, Arg-Lys, or Lys-Arg, the two latter being the most common (12), are cleaved into active peptides by specialized endoproteases, called prohormone convertases (PCs), that are responsible for most of the posttranslational processing of precursors into biologically active peptides (13). When defined in a restricted manner as serine proteases cleaving specifically at basic residues and implicated in prohormone processing, this subclass of enzymes counts at least seven members [furin, PC7, PC1/3, PC2, PACE4 (paired basic amino acid converting enzyme 4), PC5/6-A, PC5/6-B, and PC4] with distinct distribution and subcellular localization (14, 15, 16, 17). The PCs cleave the precursor at the C terminus of a basic doublet (either Arg or Lys), less often at mono- or poly basic sites (16, 17). This processing is followed by trimming of the remaining basic amino acids at the C terminus by a specific exopeptidase, called carboxypeptidase-E or -H (18). The existence of two other PC-related proteases, subtilisin/kexin isozyme-1 (19) and neural apoptosis-regulated convertase-1 (20), has been evidenced but with very different cleavage specificities.
Another enzymatic mechanism, which, so far, has retained little attention, corresponds to an inversion of the one described above: an endoprotease first cleaves at the N terminus of basic amino acids, followed by action of an aminopeptidase of the B type (21). For instance, somatostatin-28 is processed in vitro into somatostatin-14 by N-arginine dibasic convertase (NRDc) or nardilysin and aminopeptidase B (Ap-B) (22, 23, 24, 25). NRDc belongs to the M16 family of metalloendopeptidases (26), involved in various prohormone- or proprotein-processing events (27). As observed for somatostatin, dynorphin A, ANF-(1–28), or preproneurotensin-(154–170) (28, 29), the NRDc activity is restricted to the N-terminal side of either dibasic sites (28, 30), such as Arg-Arg, Arg-Lys, Lys-Arg, and Lys-Lys (24, 25), or single basic residue flanked by an aromatic or another specific hydrophobic residue (31, 32). Two isoforms of NRDc have been identified, NRD1 and NRD2; NRD2 differs from NRD1 by an insertion of 68 amino acids (corresponding to a 204-nucleotide insertion). Ap-B, a member of the M1 family of metalloexopeptidases (33), exhibits in vitro a strict selectivity toward N-terminal Arg-X and Lys-X bonds from peptides mimicking processing intermediates, as well as toward several substrates, including Arg0–Leu-enkephalin, Arg0–Met-enkephalin, and Arg–1–Lys0-somatostatin-14 (21). Like NRDc, Ap-B may be involved in propeptide- and proprotein-processing mechanisms. Nonetheless, no relevant physiological substrate of NRDc or Ap-B has yet been definitely identified.
The enzymatic mechanism implicated in the processing of MG from glucagon is still unclear despite attempts to solve this question (11, 34). The aim of our study was: 1) to characterize the MG-generating endopeptidase (MGE) activity; and 2) to precisely localize it in the -pancreatic cells, the sole cells of the organism that contain MG in a stored form. To address this issue, we first used the mouse -pancreatic TC1.6 cells, which have been shown to contain both the substrate (glucagon) and the product (MG) in the same secretory granules, as in native -cells (10), explaining why both glucagon and MG are cosecreted (10). For practical reasons, the rat pituitary GH4C1 cell line was used at various steps of our research, and rat hepatic FaO cells allowed us to compare -cell-containing MGE features with previous results obtained on liver (11).
We report here that MGE is not a PC but a set of two metalloproteases, namely NRDc and Ap-B. The rationale for this conclusion is: 1) the spectrum of sensitivity toward inhibitors clearly shows that a metalloprotease with a thiol group in the catalytic site, as well as an aminopeptidase, are involved in the MGE activity; 2) addition of recombinant NRDc in the medium enhances the activity; 3) recombinant Ap-B is able to trim in vitro N-terminal Arg residue(s) from MG-intermediate products; 4) immunodepletion of either enzyme results in the disappearance of the majority of MGE activity from the culture medium of MGE-secreting cells; 5) NRDc and Ap-B RNAs and proteins are present in the three MGE-secreting cell lines studied; 6) both proteins are found in the mature secretory granules of the -cells, close to their substrate, glucagon, and their product, MG; and 7) NRDc is present specifically in glucagon- and MG-containing fractions from perfused pancreas after physiological stimulation by hypoglycemia.
Materials and Methods
Materials
Inhibitors and cations were purchased from Calbiochem (VWR International, Fontenay-sous-Bois, France), Sigma-Aldrich (Aldrich, Saint Quentin Fallavier, France), Merck (VWR International), and Fluka (Aldrich). MG and Tyr25–glucagon-(19–25) used for RIA were synthesized in our laboratory by a solid-phase procedure, purified by HPLC and subsequent analysis of amino acid composition by mass spectrometry, as previously described (35).
Cell culture
The mouse -cells (TC1.6 cell line) were grown in DMEM containing 1 g/liter glucose, whereas the rat anterior pituitary lactotrophic GH4C1 cells and the rat hepatic FaO cells were grown in DMEM containing 4.5 g/liter glucose (DMEM, Invitrogen, Life Technologies, Cergy Pontoise, France), each supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen,) and 1% antimycotic fungizone (Invitrogen) equilibrated with 5% CO2–95% air at 37 C. Cell culture media were changed every 48 h; and, when reaching 80% of confluence, cells were detached with 2.5% trypsin, 0.5 mM EDTA (Invitrogen).
Enzyme Assays
The MGE activity was obtained from conditioned media of all cell lines after repetitive 2-h incubation periods at 37 C without serum. After collection of the media, two other incubations were performed in the same way, and pooled. The resulting cell-conditioned media were then concentrated approximately 300-fold by centrifugation on Centricon (Amicon, Millipore Corp., Bedford, MA), as previously described (36). Enzymatic preparation was stored at –20 C in 30% glycerol before enzymatic assays. All assays were performed in triplicate, using synthetic mammalian glucagon as substrate (50 pmol, i.e. final concentration of 200 nM), in a final vol of 250 μl in 50 mM Tris acetate/2 mM CaCl2 buffer (pH 7.5). All reaction mixtures were prepared at 4 C, preincubated at 37 C for 15 min with or without different inhibitors, and were incubated for 1 h with glucagon. All reactions were stopped by heating the samples at 100 C for 5 min. The amount of MG produced was measured by RIA.
RIA of MG
As previously described (37, 38), we used mono 125I-Tyr25–glucagon-(19–25) as the tracer, Nle29-glucagon-(19–29) as the standard, and a rabbit anti-MG serum named SAR (37), which specifically recognizes the N-terminal epitope present in MG and masked in glucagon, the mother-molecule (the cross-reaction of glucagon in the assay is about 0.2%). We evaluated the MGE activity by reference to the control assay in which the enzyme was incubated for 1 h at 37 C with glucagon, at pH 7.5. In the presence of different inhibitors, the observed values were computed with reference to the control assay considered as 100%. Results are presented as means ± SEM of at least three experiments.
RT-PCR
Total RNA was extracted from each cell line by the RNAzol method (Extract All, Eurobio, Les Ulis, France). Equal amounts of total RNA (1 μg) from each sample were treated with DNase I (Amplification Grade, Invitrogen) and reverse-transcribed at 42 C using Superscript II Reverse Transcriptase (Invitrogen). To amplify specific regions of Ap-B and NRDc cDNAs, sequence-specific primers were designed on the basis of their nucleotide sequences obtained from GenBank. After an initial denaturation step at 94 C (3 min), thirty cycles of PCR were carried out at 94 C (30 sec), 62 C (30 sec), and 72 C (1 min) in a robocycler gradient 96 (Stratagene Ltd., Cambridge, UK) using Taq polymerase (Roche Diagnostics, Meylan, France). Sense and antisense primer sequences used to amplify rat Ap-B cDNA template (Accession number U61696) were 5'-ACC TTT GTC ACC CCG TGC CTG CTA-3' and 5'-GGG TTT TCC TCT CCA GAC ACA TCC-3', respectively. These specific primers were designed to produce a 233-bp-long DNA fragment. For mouse Ap-B cDNA template (39), sense and antisense primers were 5'-CAA GGA GGA ATA CAG TGG GGT CAT-3' and 5'-CAT TGT AGG TGT CAT CGG GGT CCA-3', respectively. These primers led to the production of a 431-bp-long DNA amplification product.
For rat NRDc cDNA template (Accession number L27124), sense and antisense primers were 5'-GTG GAG GGT AAA ACA GGA AAT GCA-3' and 5'-AGG CAT CAA ATC CAT TCT CAT CTG-3', respectively. The rat 5' primers were chosen 187 bp upstream from the 204 nucleotides insertion (corresponding to the 68 amino acids coding sequence), present only in the NRD2 isoform, to identify which RNAs are present in this cell (40). These primers discriminate between both NRDc isoforms (NRD1 and NRD2), leading to the production of two different fragments of 385 and 589 bp in length, respectively. For mouse NRDc (accession number U86112), primers were 5'-CGT TCT TCT CTC TGG TGC TAA TGA-3' and 5'-ATA GGC GGG TTC GGC AAG GTT GTG-3', respectively, resulting in an amplification product 373 bp long. Amplified RT-PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining.
Western blotting
Total proteins were extracted from cells grown in 10-cm culture dishes with 1 ml lysis buffer (50 mM HEPES, 1% Triton X-100, 1 mM EDTA, 30 mM pyrophosphate, 1 mM vanadate, 1 mM PMSF, 1 mg/ml bacitracin) for 30 min at 4 C. Cell lysates were then centrifuged for 30 min at 4 C at 14,000 rpm. The protein concentration of the collected supernatant was determined by Bradford miniassay. For Western blot analysis, proteins were denatured by boiling during 5 min in Laemmli buffer, separated on a 5% or 7.5% sodium dodecyl sulfate-polyacrylamide gel for the identification of NRDc or Ap-B, respectively, and transferred on a nitrocellulose membrane. After blocking overnight at 4 C in PBS containing 0.1% Tween 20 and 5% BSA or 10% of milk blocking buffer for NRDc and Ap-B, respectively, membranes were probed with rabbit polyclonal anti-NRDc serum (diluted 1:5000) directed against the acidic domain of NRDc (29, 41), or with a rabbit polyclonal anti-Ap-B serum (diluted 1:2000) directed against purified Ap-B (21), in blocking buffer for 1 h at room temperature. The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (diluted 1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA). The antigen-antibody complexes were visualized using the ECL chemiluminescence detection kit (Amersham Life Sciences, Saclay, France) and observed with a Kodak Image Station 2000 system (Eastman Kodak Co., Rochester, NY).
Isolation of islets of Langerhans
Islets were isolated from adult male Wistar rats using collagenase digestion and collected after centrifugation on a Ficoll density gradient as previously described (10). For electron microscopy studies, islets were incubated for 1 h at 37 C in Krebs-Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H20, 10 mM NaHCO3, pH 7.4) containing 1 g/liter BSA and 2.8 mmol/liter glucose. For immunofluorescence experiments, islets were cultured in RPMI-1640 containing 5.6 mmol/liter glucose, 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/liter glutamine for 24 h.
Immunofluorescence
After dissociation with 0.025% trypsin/0.01% EDTA, islets were seeded on poly-L-lysine-coated-(Sigma-Aldrich) LabTek Chamber Slide System. After a 7-d culture period, they were fixed with 2% paraformaldehyde in PBS for 20 min and permeabilized by a 5-min incubation in 0.1% Triton X-100. After saturation with 2% BSA solution for 20 min, cells were incubated overnight with the same Ap-B or NRDc antiserum used for Western blotting (diluted 1:50) and a sheep antiglucagon antibody (diluted 1:100) directed against centro/N-terminal glucagon (Biogenesis, Poole, UK). After washing, a fluorescein isothiocyanate-conjugated rabbit antisheep (Vector Laboratories, Burlingame, CA) and a Texas Red-conjugated goat antirabbit antibody (Vector Laboratories) were applied separately to the cells for 1 h (both diluted 1:100). Cells were finally mounted in Citifluor (Citifluor LTD, London, UK) and observed with a Bio-Rad (Hercules, CA) confocal microscope equipped with an argon-krypton laser using the facilities of the Centre Régional d’Imagerie Cellulaire (Montpellier, France). The negative control was performed by incubating the cells only with the secondary antibody.
Electron microscopy
Isolated islets were fixed with 2.5% paraformaldehyde/0.1% glutaraldehyde in 100 mmol/liter phosphate buffer (pH 7) for 1 h at room temperature. The islets were then washed in 50 mmol/liter NH4Cl-PBS, postfixed in 1% osmium tetroxide for 2 min, dehydrated in an ascending series of ethanol, and embedded in LR White (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin (60 nm) sections were cut using a Reichert ultramicrotome (Ultracut S, Vienna, Austria) and deposited on gold grids. Sections were saturated with 10% fetal calf serum in PBS and incubated overnight at room temperature with a rabbit antiglucagon, anti-Ap-B, or anti-NRDc serum (diluted 1:100, 1:20, and 1:50, respectively), all produced in our laboratories. After washing, an antirabbit antibody (diluted 1:25) labeled with 10-nm gold particles (British Biocell, Cardiff, UK) was applied to the sections for 1 h. Washed sections were finally stained with 2% uranyl acetate for 20 min and observed with a transmission electron microscope (Hitachi H-7100, Hitachi, Düsseldorf, Germany). The specificity of the immune reaction, for both immunofluorescence and electron microscopy, was tested by incubating the sections only with the secondary antibody.
Reverse-phase HPLC
The concentrated supernatant of GH4C1 cells was incubated for 1 h at 37 C with 20 nmol glucagon as substrate in 50 mM Tris acetate/2 mM CaCl2 buffer (pH 7.5), 100 μM bacitracin (to avoid degradation of the peptide produced), and with 1,10-phenanthrolin (1 mM) or amastatin (500 μM). Control was performed with similar incubation contents without inhibitor. Synthetic peptides corresponding to expected intermediate products containing one (R-MG) or two (RR-MG) arginine extensions and the final product, MG, were run on HPLC to calibrate the column. The incubation medium containing the resulting cleavage products was applied to an Uptisphere silica ODB column (Interchim, Montlu?on, France) at room temperature. The column solvent was water + TFA 0.05% (solvent A) for aqueous phase and acetonitrile (solvent B) for the organic phase. The HPLC system was operated at 1 ml/min and 25% of solvent B. Elution was conducted with a linear gradient of 25–40% of solvent B for 40 min at 1 ml/min. Incubation with purified rat Ap-B, was performed for 1 h at 37 C with 1 nmol of processing intermediates (RR-MG or R-MG) as substrates in 0.1 M borate buffer, containing 150 mM NaCl (pH 7.2). The cleavage products were separated by HPLC using the protocol described above.
Immunoprecipitation procedure
Concentrated supernatants from TC1.6 were incubated with a primary antibody directed against NRDc (diluted 1:200) or Ap-B (diluted 1:500) overnight at 4 C. Immunocomplexes were then precipitated from the supernatant with protein A/G plus-Agarose (Santa Cruz Biotechnology) for 4 h at 4 C. After a 5-min centrifugation at 3000 rpm, the supernatants were collected. Standard reaction assays were performed using 50 pmol glucagon incubated for 2 h at 37 C. Reactions were stopped by boiling at 100 C, and the MG produced was assayed by RIA, as described above. Controls were performed by omitting the primary antibody or by using an unrelated primary antibody used for immunoprecipitation of the PI3-kinase catalytic subunit (Tebu-bio s.a, Le Perray en Yvelines, France).
Isolated perfused rat pancreas fractions
Fractions from rat isolated perfused pancreas obtained as described (10) were concentrated by lyophilization and analyzed for their content in NRDc by Western blotting after immunoprecipitation of the enzyme, both procedures being performed according to the above Western blotting and Immunoprecipitation procedure sections.
Statistical analysis
All results are presented as mean values ± SEM together with the number of individual determinations. Results were expressed as mean ± SEM and data compared using the Student’s t test. Groups of data were considered significant at P < 0.01 (**) or P < 0.05 (*).
Results
From the first attempts to isolate the enzymatic system (MGE) responsible for the secondary processing of glucagon into MG (11, 34, 38), it appeared that the highly purified MGE from rat liver membranes (24, 38) had the features of a metalloprotease with a requirement for a free thiol group in the catalytic site. Because the single cell in the organism that stores and secretes MG is the glucagon-secreting -cell of the islets of Langerhans, we studied the presence of an activity of the MGE type in the conditioned medium of the TC1.6 cell line. The rationale for using this cell line was: 1) these cells are well differentiated with features close to that of the native -cells of the islets of Langerhans; in particular, they are known to store and secrete glucagon and MG (10); and 2) we had direct access, via the culture medium of this cell line, to the secreted products in which we expected to find the enzymatic system producing MG, which was shown to be present in the same secretory granules as glucagon. From that point, we started to characterize the MGE activity present in the conditioned medium of the TC1.6 cell line. We extended our observations to a hepatic cell line (FaO), which represents the equivalent in culture of the hepatic cells in which we had described MGE originally, and to the GH4C1 cell line, an apparently not physiologically relevant cell line of pituitary origin but with an endocrine and highly transformed character (unlike the TC1.6), which makes it a suitable source for running experiments which necessitate large amount of material.
Effects of proteinase inhibitors on the MGE activity released from the TC1.6 cell line
After having established the presence of a MGE activity in the concentrated culture medium of TC1.6 cells, we analyzed its enzymatic features using various proteinase inhibitors. The results were compared with previous data obtained with purified MGE from rat liver plasma membranes (11, 34, 38).
As shown in Table 1, we observed a modest, yet significant, inhibitory action of EDTA, a profound inhibition induced by the metal chelator 1,10-phenantrolin and clear-cut effects of thiol reagents, in particular dithiothreitol (DTT). We conclude that the enzymatic system producing MG from glucagon in -cells shares, with that present in liver, a thiol-dependent metalloendoprotease character, both exhibiting a similar inhibitory profile (11, 38). Moreover, the MGE activity present in hepatic FaO cells shares similar enzymatic features toward EDTA, 1,10-phenantrolin, and DTT (about 25%, 7%, and 20% remaining MGE activity at the highest concentration, respectively; data not shown). In addition, the aminopeptidase inhibitors amastatin and bestatin inhibited the MGE activity at low doses, suggesting the involvement of an aminopeptidase activity in the maturation process present in TC1.6 cells (Table 1), as well as in FaO cells (around 52% and 60% at the highest concentration, respectively; data not shown).
TABLE 1. Comparison of proteinase inhibitors effects on the MGE activity released from the TC1.6 cell line
Effects of zinc, cobalt and calcium ions on MGE activity from TC1.6 cells and its restoration by metallic ions after extinction by 1,10-phenanthrolin
The apparent metalloprotease character of MGE led us to test the effect of zinc, cobalt, and calcium ions on the MGE activity. As shown in Table 2, the addition of zinc or cobalt cations, known to control the catalytic activity of metalloendoproteases, dose-dependently inhibited the MGE activity secreted by the TC1.6 cells; whereas calcium, necessary for a PC activity, had no effect on MG production. This strongly supports the metallopeptidase character of MGE, which is fully consistent with previous data on the MGE activity obtained from hepatic plasma membranes (11, 34, 38), as well as from hepatic FaO cells, similarly affected by zinc and cobalt addition (data not shown).
TABLE 2. Effects of zinc, cobalt and calcium ions on the MGE activity from TC1.6 cells and its cation reactivation profile after metal chelation by 1,10-phenanthrolin
Furthermore, after its suppression by the metal chelator 1,10-phenanthrolin, the MGE activity was restored by addition of zinc or cobalt, with a blunting effect at higher concentrations, with, again, no effect of the calcium ion.
These observations fit well with the known sensitivity of metalloendoproteases toward the metallic ion implicated in the catalytic activity of such enzymes (24), and rules out the implication of a PC, which belong to a family of calcium-dependent serine endoproteases (16, 17). The sensitivity toward the metal ions, both in their suppressing effect and in their restoration capabilities, was very similar for MGE obtained from FaO cells, again demonstrating the presence of the same enzymatic system in both tissues.
Ap-B, potential component of MGE, is able to cleave intermediate products of glucagon processing
In view of the effects of the aminopeptidase inhibitors amastatin and bestatin, we have hypothesized that MGE cleaves glucagon sequentially, including a first cut by a metalloendoprotease at the level of the basic doublet followed by trimming of the basic amino acid(s) by an aminopeptidase of the B type (aminopeptidase-B or Ap-B).
From our studies in various cell lines, it appeared that an abundant MGE activity was present in the conditioned medium of the pituitary GH4C1 cells, with all the enzymatic features found in the media from TC1.6 and FaO cells (data not shown). According to the apparent identity of MGE from the three cell lines and considering the abundance of MGE activity in the supernatant of GH4C1 cells, we have used the supernatant of those cells as the MGE archetype at that step of the work. The concentrated culture medium of GH4C1 cells was incubated with glucagon for 1 h at 37 C, with or without amastatin or 1,10-phenanthrolin at concentrations observed to inhibit MGE activity, and run on HPLC after column calibration using synthetic peptides corresponding to expected intermediate products (R-MG or RR-MG) and the final product, MG. As shown in Table 3A, we observed the presence of the three peptides (RR-MG, R-MG, and MG). Addition of amastatin resulted in the complete disappearance of MG, whereas the amounts of intermediate products, RR-MG and R-MG, increased. On the other hand, the production of RR-MG, R-MG, and MG was completely suppressed by addition of 1,10-phenanthrolin (Table 3A). These data strongly suggest that, in accordance with our previous working hypothesis, glucagon is processed into MG by a metalloendoprotease cleaving the substrate at the level of the Arg17–Arg18 site, followed by an aminopeptidase, which trims the remaining arginine residue(s), leading to the final product, MG.
TABLE 3. Proportion of glucagon intermediate products after incubation of GH4C1 MGE, NRDc or recombinant Ap-B activities with glucagon or other substrates
To further establish the existence of this sequential cleavage, recombinant Ap-B was incubated with its potential substrates RR-MG and R-MG (Fig. 1). This resulted in MG production using R-MG as substrate (Fig. 1B and Table 3B), whereas incubation with RR-MG led to R-MG and MG production (Fig. 1C and Table 3B). Furthermore, addition of amastatin reduced the yield of MG, whatever the Ap-B substrate (Table 3B). It is interesting to note that Ap-B was unable to cleave in vitro the basic doublet of glucagon (Table 3B), indicating the absence of endoproteolytic activity of Ap-B, and thus that a metalloendoprotease is necessary, upstream of the aminopeptidase, in the MGE activity. Addition of a double amount of Ap-B resulted in the complete transformation of R-MG into MG (data not shown).
FIG. 1. HPLC profiles of peptides produced after incubation of recombinant Ap-B with potential intermediate products from NRDc activity during the course of in vitro glucagon to MG processing. A, Control run with enzymatic preparation alone. B, Production of MG from R-MG used as the substrate. C, Production of MG and the intermediate peptide (R-MG) from RR-MG used as the substrate.
These results are in full agreement with the hypothesis that Ap-B is responsible for trimming the basic amino acids after the action of an endoprotease on the Arg-Arg doublet of glucagon.
NRDc, potential component of MGE, is able to process glucagon into MG
Because a primary endoproteolytic mechanism is necessary to release Ap-B substrates and because such a sequential cleavage, involving NRDc/Nardilysin and Ap-B metalloproteases, has already been identified in the processing of somatostatin-28 into somatostatin-14 (22, 23, 24, 25), we have hypothesized that the endoprotease might be NRDc. To address this issue, recombinant NRDc was added to the medium from GH4C1 cells (containing endogenous Ap-B, necessary to uncover the MG epitope for MG RIA), and MG production was evaluated and compared with its production by the MGE from GH4C1 alone.
As shown in Table 3C, addition of recombinant NRDc to the GH4C1 medium significantly increases the MG production, although to a low extent (see Discussion).
Altogether, these data are fully compatible with our hypothesis that MGE is composed of a set of two metalloproteases: NRDc and Ap-B.
The MGE activity is inhibited after immunoprecipitation of NRDc or Ap-B from a crude MGE preparation
In an attempt to confirm our preceding results, we used another technical approach, i.e. immunoprecipitation of either the NRDc or the Ap-B from the MGE-containing conditioned medium from our cell lines by using specific antibodies. The immunodepleted culture medium was incubated with the substrate glucagon, and the remaining activity was measured. As shown in Fig. 2A, a majority of the MGE activity was removed by the immunoprecipitation procedure, with a 85.3% and 85.2% reduction from the GH4C1 cells, using the anti-NRDc and the anti-Ap-B antibodies, respectively.
FIG. 2. Effect of immunodepletion of NRDc or Ap-B on MG production from GH4C1 or TC1.6 MGE. A, After immunoprecipitation of NRDc or Ap-B using the corresponding specific antibody (NRDc or Ap-B) or without antibody (control, noted CTL) from the GH4C1 cells supernatant containing MGE activity, the supernatant was incubated with glucagon as the substrate for 2 h at 37 C. MG production was measured by MG RIA. B, Similar immunoprecipitations were performed using the supernatant from the TC1.6 cells, and the same experiments were carried out. The results represent means ± SEM from at least three separate experiments. * or **, Significantly different from the control (P < 0.05 or P < 0.01, respectively; Student’s t test). C, As a negative control, we used, on the MGE preparation from TC1.6 cells, an unrelated antibody (Ab) against the PI3-kinase catalytic subunit to exclude any spurious effect in the immunoprecipitation experiment.
A similar profile was obtained for the MGE activity from TC1.6 cells (Fig. 2B): the activity was inhibited by 86% or 87.3%, with anti-NDRc and anti-Ap-B antibodies, respectively. As a negative control, we used an unrelated antibody, i.e. a PI3-kinase catalytic subunit-directed antibody, known to be active in separate PI3-kinase immunoprecipitation experiments. No decrease in the MGE activity was noted (Fig. 2C), ruling out any spurious effect of the anti-NDRc or the anti-Ap-B antibody.
These results strongly strengthen precedent data and clearly indicate that both NRDc and Ap-B are deeply involved in the MGE activity, by acting sequentially to release MG from the mother-hormone.
NRDc and Ap-B, the two potential components of MGE, are present in MG-producing tissues
Our results led us to conclude that the MGE activity responsible for the in vitro processing of glucagon into MG is composed of both NRDc and Ap-B. However, neither the expression nor the subcellular localization of NRDc and Ap-B has yet been established in these cell lines. To address this issue, we used four complementary approaches: RT-PCR, Western blotting, immunofluorescence, and electron microscopy.
By RT-PCR, we identified amplified PCR products of the rat and mouse Ap-B at the expected size (Fig. 3, A and B). As described in Materials and Methods, two alternatively spliced isoforms of NRDc could be detected in rat cell lines (rNRD1 and rNRD2; 385 bp and 589 bp amplification bands, respectively) (Fig. 3A), whereas a single amplification band of 373 bp for the mouse NRDc could be detected in the TC1.6 cell line (Fig. 3B). The PCR products were analyzed by sequencing the corresponding gel-purified bands (data not shown). The main isoform of NRDc present in rat cells is NRD1, whereas a faint band corresponding to NRD2 was visible in both rat cell lines (Fig. 3A). In addition, we observed some other RT-PCR bands in the rat FaO cell line (Fig. 3A, left), but the scarcity of the material precluded any further identification.
FIG. 3. Presence of both NRDc and Ap-B RNAs and proteins in GH4C1, TC1.6, and FaO cells. A, Results from PCR after reverse-transcription of RNAs from GH4C1 and FaO cell lines, by using specific primers (see Materials and Methods). Each PCR product was visualized with ethidium bromide. Lanes 1 and 3, Rat Ap-B primers; lanes 2 and 4, rat NRDc primers. The product lengths were 385 bp or 589 bp and 233 bp, respectively for NRD1 or NRD2 and Ap-B-amplified products. B, RT-PCR of mouse TC1.6 cDNAs. Lane 1, Mouse Ap-B primers; lane 2, mouse NRDc primers. The product lengths were 431 bp and 373 bp for Ap-B and NRDc amplified products, respectively. C, Western blot analysis of Ap-B in GH4C1, FaO, and TC1.6 cell lines. Total cell lysates (35 μg) were processed on 7.5% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with the specific antibodies against Ap-B. D, Similar protein extracts were separated on a 5% polyacrylamide gel and incubated after transfer with the specific antibodies against NRDc. Partially purified NRDc and Ap-B proteins were used as controls. Standard proteins (molecular mass markers, in kDa) are indicated along the blot.
Using specific antibodies developed against purified Ap-B (21) or against the acidic stretch of NRDc (41), we show the presence of both proteins by Western blotting in the different cell lines. Indeed, the GH4C1 and FaO cell lines contain the 72-kDa Ap-B form (Fig. 3C, lanes 1 and 2, respectively). In TC1.6 cells (Fig. 3C, lane 3), we observed a band of weaker intensity with the same migration profile as the Ap-B standard (Fig. 3C, lane 4) and those from rat cell lines (Fig. 3C, lanes 1 and 2).
In Fig. 3D, we observed the presence of a major band (140 kDa) corresponding to NRD1 (Fig. 3D, lanes 1–3), with a migration profile similar to that of the NRDc control (Fig. 3D, lane 4). We also observed an upper band corresponding to NRD2 (145 kDa), with a weaker intensity, suggesting its lower expression in the rat cell lines, whereas it was undetectable in the TC1.6 cells. These results are in line with the presence in the rat cell lines of the 385 bp as the major amplicon corresponding to NRD1 and the minor 589-bp amplicon corresponding to NRD2 (Fig. 3A).
Hence, our results show the expression of mRNAs coding for both NRDc and Ap-B, as well as their translation products in the glucagon- and MG-producing -cells.
NRDc and Ap-B are colocalized with their substrate glucagon in the -cells
In immunofluorescence confocal microscopy studies (Fig. 4), glucagon staining appears to be granular in -cells freshly isolated from rat islets of Langerhans, the punctuated staining corresponding to glucagon-containing secretory granules (Fig. 4, A and D). The Ap-B staining, also granular, appears to be similar to that observed for glucagon (Fig. 4B).
FIG. 4. Localization of glucagon and Ap-B or NRDc by double immunofluorescence. Dissociated pancreatic islets, cultured for 7 d on LabTek slides, were fixed with 2% paraformaldehyde, followed by immunoreaction. Pancreatic -cells were double-immunostained with antiglucagon (A and D) and anti-Ap-B (B) or anti-NRDc (E) antibodies. Immunofluorescence was analyzed by a dual-channel confocal microscope. Glucagon and Ap-B or NRDc stainings appear as green (A and D) and red (B and E), respectively. Coincidence of both fluorescences results in the appearance of a yellow color (C and F). Pictures G and H represent the double staining of TC1.6 cells with antiglucagon and anti-Ap-B or antiglucagon and anti-NRDc antibodies, respectively. Picture I is a negative control (omission of the primary antibody). Scale bars, 10 μM.
Similar observations were made for NRDc (Fig. 4E) with the antibody used for Western blotting and directed against the acidic stretch domain of NRDc. Coincidence of the two fluorescences shows that both enzymes obviously colocalize (Fig. 4, C and F) with glucagon in normal -cells isolated from rat islets of Langerhans. The staining appeared as discrete, punctuated, which is typical for a granular staining, observed for a secretory vesicle localization. Together with the already known colocalization of glucagon and MG in the granules (10), this observation demonstrates that the substrate (glucagon), the product (MG), and the processing enzymes colocalize in secretory granules and strongly supports our hypothesis.
As shown in Fig. 4, G and H, respectively, a similar Ap-B/glucagon or NRDc/glucagon colocalization was observed in the glucagon-secreting TC1.6 cells. Figure 4I demonstrates the specificity of the labeling that disappeared when the first antibody was omitted.
Even more precise evidences for the colocalization of the substrate glucagon and the processing enzymes were obtained using gold particles immunostaining at the ultrastructural level (Fig. 5). Indeed, both proteins were detected at the periphery of the mature glucagon secretory granules, again in line with the already observed MG at that place (10). These results further demonstrate the presence of Ap-B and NRDc in glucagon secretory vesicles of the -cells, at the periphery of mature glucagon-containing granules. In addition to its presence in secretory granules, NRDc could also be observed at cytosolic locations.
FIG. 5. Subcellular localization of Ap-B and NRDc in pancreatic -cells of rat islets by immunogold electron microscopy. Electron microscopy was performed on ultrathin sections of isolated islets from rat pancreas after immunostaining according to the gold particles technique as described in Materials and Methods. The figures represent part of the cytoplasm of pancreatic -cells, rich in secretory granules. Staining was performed using rabbit antiglucagon (upper panel), rabbit anti-Ap-B (middle) or rabbit anti-NRDc (lower) antibodies, followed by secondary antibody coupled with 10-nm gold particles (directed against rabbit antibodies. Magnification, x80,000.
NRDc, the major component of MGE, is cosecreted with glucagon and MG
To further ascertain that NRDc is present in the secretory pathway, in contrast to what is usually thought, and is secreted together with the substrate glucagon and the product MG, we selected preserved fractions of the experiments that allowed us to demonstrate cosecretion of glucagon and MG (10). As shown in Fig. 6, neither the insulin-containing 11-mM fraction immediately before the switch to 2.8 mM nor the fraction collected 2 min after this switch, which do not contain significant amounts of glucagon or MG (10), show any detectable amount of NRDc, using the immunoprecipitation procedure followed by Western blotting. In contrast, two fractions selected close to the secretion peak of glucagon and MG [7 and 12 min of hypoglycemic stimulation of glucagon and MG secretion (see Ref.10)] clearly contain NRDc (Fig. 6). This proves that, upon physiological stimulation of glucagon and MG secretion by decreasing the glucose concentration, NRDc is cosecreted with both peptides and thus is present in the regulated secretory pathway. As expected, similar results were obtained using an Ap-B antibody, confirming the presence of Ap-B together with NRDc in glucagon- and MG-containing fractions (data not shown).
FIG. 6. Presence of NRDc in glucagon- and MG-containing fractions from perfused rat pancreas after physiological stimulation by hypoglycemia. Selected fractions from isolated perfused rat pancreas (10 ) (see Materials and Methods) were analyzed, after NRDc immunoprecipitation, by Western blotting for their NRDc content. 11 mM, Fraction at the end of the insulin-stimulatory period, which does not contain either glucagon or MG (10 ). The three next fractions correspond to 2, 7, and 12 min after glucagon (and MG) stimulation period by hypoglycemia (2.8 mM glucose). Fractions 7 and 12 were selected close to the secretion peak of glucagon and MG.
Discussion
MGE, responsible for the secondary processing of the mother-hormone glucagon into MG (11, 34, 38), controls the glucagon/MG balance, giving this enzyme a pivotal role in this new level in the regulation of metabolism. In the present report, we characterize the glucagon-processing enzyme contained in MG-producing tissues and demonstrate that MGE is made of two metalloproteases, NRDc and Ap-B, which act sequentially, leading to the release of the final active product, MG. We have also identified their cellular and subcellular localization in the -cells of islets of Langerhans, where both enzymes are colocalized in mature glucagon-containing secretory granules, beside their substrate glucagon, as well as the product, MG, and are secreted together with the two peptides.
Our data yield new insights into prohormone processing, classically initiated in the trans-Golgi network by one or several specific endoproteases of the PC type at monobasic, dibasic, and/or multibasic residues (12, 13, 16, 17). Our data fit with the fact that the secondary processing of glucagon into MG is distal to those of proglucagon, which occurs, in -cells, via action of prohormone convertase-2 (42) and thus should be considered as an additional level of prohormone processing, glucagon being considered, in that case, as a prohormone. The metallic ion sensitivity of MGE (notably for zinc and calcium) is consistent with a metalloendoprotease activity (26) and excludes the involvement of a calcium-dependent serine endoprotease of the PC type (16, 17). A similar cleavage scheme has already been shown for the in vitro processing of somatostatin-28 into somatostatin-14, involving NRDc and Ap-B (22, 23, 24, 25). Concerning this question, the evidence that no somatostatin-14 was observed in pancreatic -cells or in brain from PC2-null mice (43, 44) does not rule out the implication of the NRDc and Ap-B system in some tissues, because there is evidence that prosomatostatin processing is not uniform (45) and that prosomatostatin processing up to somatostatin-14 necessitates, in some tissues, a somatostatin-28 step which appears to be PC2 dependent (43, 44, 45).
The involvement of Ap-B in prohormone processing has already been shown to occur either in the secretory pathway, at the plasma membrane, or at both localizations (46). This ubiquitous enzyme, present in endocrine and nonendocrine cells (22, 47) and associated with secretory vesicle membranes from cattle pituitary (46), neurons, and PC12 (47), was described to be secreted both via the constitutive and the regulated pathways (21, 33, 47).
Besides, NRDc localization is more puzzling. The enzyme has been found mainly localized in cytoplasm with significant amounts associated with microtubular structures and secreted by some cell types (48). NRDc activity, present in culture media, has also been found in other intracellular compartments among which secretory granules (49). Although controversial, NRDc was supposed, as shown for PCs, to mature prohormones at dibasic sites within the secretory pathway in cytoplasm and/or at the cell surface (50, 51). This assumption, despite the lack of definite function for the enzyme, was reinforced by the presence of two putative N-glycosylation sites together with a modest putative signal peptide. Besides, a Met49 NRDc isoform, translated from the second initiation codon, has been observed in rat testis (52); the lack of signal peptide in that isoform excluded a priori its potential involvement in prohormones processing in secretory granules of endocrine cells (41, 52). These discrepancies may be explained by a tissue-specific distribution of NRDc isoforms, with a possible expression of Met1 NRDc (containing the signal peptide) in pancreatic -cells and not in other endocrine cells. Because our immunohistochemical experiments were performed using an antiacidic domain of NRDc antibody, we could not discriminate between Met1 and Met49 NRDc isoforms. Whatever the molecular explanation, our data using both morphological and biochemical approaches demonstrate that both enzymes are present in the regulated secretory pathway, at least in -cells.
Interestingly, different molecular forms of cathepsin L have recently been implicated in the production of (Met)enkephalin from its precursor, and its presence demonstrated in secretory vesicles (53). Similarly to the predicted cleavage specificity of MGE, the cathepsin L isoform present in secretory vesicles requires the presence in the intermediate products of N-terminal basic extensions later trimmed by an Ap-B-like activity (53, 54). The present data are thus compatible with the hypothesis that the isoform present in -cells differs from that present in rat testis, or that a chaperone protein retains Met49 NRDc at the periphery of secretory granules. On the other hand, glucagon is as a rather poor substrate for NRDc in vitro (Prat, A., unpublished, Table 3C), when compared with other known substrates such as dynorphin A or somatostatin (28, 29). This might, at a first sight, appear as incompatible with a physiological relevance of the proposed mechanism. This apparently poor adequacy between the enzyme and the substrate is certainly meaningful in this particular case, because only a marginal portion (3–5%) of glucagon is transformed in vivo into MG in rat -cells (10), as well as in humans (55) and at a similar rate for circulating glucagon in target tissues, thanks to the presence of MGE at the cell surface (8, 11, 34, 38). This partial processing is, on the contrary, particularly relevant from a physiological point of view (10), because it fits with the fact that MG (active in the subpicomolar range) is at least three orders of magnitude more potent than glucagon (active in the nanomolar range) on target tissues. In addition, it should be considered that a very efficient processing of glucagon into MG, by antagonizing totally the metabolic effects of the mother-hormone would be antinomic, regarding the counter-regulatory hormone status of glucagon. A recent study (56) has described the production of a small amount of MG among 13 different degradation products, probably by a 170-kDa serine protease; this process observed in hepatic endosomes corresponds to the degradation of internalized glucagon and differs entirely from the specific processing mechanism described here.
MG generated by the MGE activity is now recognized as an original peptide involved in novel regulatory mechanisms. Indeed, in contrast to its mother-hormone, glucagon, it inhibits locally insulin release within the islets of Langerhans (7, 10), whereas its production from circulating glucagon in target tissues (8, 9, 11) allows a new level of regulation, again in a direction opposite to that of the mother-hormone. Moreover, MG has been recently shown to reduce insulinemia in vivo, whereas it favors the peripheral action of insulin (57). This confers to the small peptide the status of a new entity in the regulation of metabolism. Thus, the ability of MGE to produce from glucagon a factor which negatively modulates pancreatic and peripheral effects of the mother-hormone is, from a physiological point of view, of great interest. Accordingly, the glucagon/MG balance, at the origin of which is MGE, is a new parameter of potential importance to fully understand the molecular and cellular bases of fuel homeostasis and their impairment in metabolic diseases.
The present report provides an essential clue that was missing for a full understanding of the physiological pathways in which the glucagon/MG balance is involved. The two components of MGE being now identified, it will be possible by modulating their expression in vitro (small interfering RNA, overexpression) or in vivo (knock-out, knock-in) to analyze the importance of MG production in the biological regulation as well as to obtain information on its possible role in pathological states, such as type 2 diabetes.
Acknowledgments
We are grateful to Christine Fahy (Centre National de la Recherche Scientifique Unité Mixte de Recherche 7631, Université Pierre et Marie Curie, 75006 Paris, France) for technical assistance in HPLC procedures and Antoine Disset (Centre National de la Recherche Scientifique Unité Propre de Recherche 1142, Montpellier, France) for sequencing the purified PCR products.
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Address all correspondence and requests for reprints to: Dominique Bataille, Institut National de la Santé et de la Recherche Médicale Unité 376, Centre Hospitalier Universitaire Arnaud de Villeneuve, 371, Rue du Doyen G. Giraud, 34295 Montpellier, Cedex 5, France. E-mail: bataille@montp.inserm.fr.
Abstract
Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17–Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing -cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting TC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse TC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the TC1.6 and native rat -cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of -cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
Introduction
A WIDE VARIETY of peptidic hormones and regulatory peptides are produced from larger proteins, called prohormones (1). A typical example of a prohormone is proglucagon, a 160-amino-acid peptide from which originate several hormones with different biological roles (2, 3, 4) through tissue-specific posttranslational processing. In the -cells, the 29-amino-acid peptide glucagon is its major end product (5), but a secondary posttranslational processing occurs that leads to the C-terminal (19–29)-fragment of glucagon, called miniglucagon (MG) (6, 7). This peptide displays original properties opposite to those of glucagon, the mother-hormone; among them figure a negative inotropic action on embryonic chick cardiac cells (8), inhibition of the liver plasma membrane calcium pump (9), and interestingly, an inhibitory effect on secretagogue-induced insulin secretion from ?-cells (7, 10). The posttranslational processing at the Arg17–Arg18 dibasic site of glucagon leading to MG was demonstrated to occur in the glucagon-producing -cells of the islets of Langerhans (10) but also from circulating glucagon in heart and liver (8, 11). In both cases, the small proportion of MG produced is more than enough to be biologically relevant, because of the huge potency of MG compared with glucagon (7). Classically, precursors containing basic residues arranged in doublets, such as Lys-Lys, Arg-Arg, Arg-Lys, or Lys-Arg, the two latter being the most common (12), are cleaved into active peptides by specialized endoproteases, called prohormone convertases (PCs), that are responsible for most of the posttranslational processing of precursors into biologically active peptides (13). When defined in a restricted manner as serine proteases cleaving specifically at basic residues and implicated in prohormone processing, this subclass of enzymes counts at least seven members [furin, PC7, PC1/3, PC2, PACE4 (paired basic amino acid converting enzyme 4), PC5/6-A, PC5/6-B, and PC4] with distinct distribution and subcellular localization (14, 15, 16, 17). The PCs cleave the precursor at the C terminus of a basic doublet (either Arg or Lys), less often at mono- or poly basic sites (16, 17). This processing is followed by trimming of the remaining basic amino acids at the C terminus by a specific exopeptidase, called carboxypeptidase-E or -H (18). The existence of two other PC-related proteases, subtilisin/kexin isozyme-1 (19) and neural apoptosis-regulated convertase-1 (20), has been evidenced but with very different cleavage specificities.
Another enzymatic mechanism, which, so far, has retained little attention, corresponds to an inversion of the one described above: an endoprotease first cleaves at the N terminus of basic amino acids, followed by action of an aminopeptidase of the B type (21). For instance, somatostatin-28 is processed in vitro into somatostatin-14 by N-arginine dibasic convertase (NRDc) or nardilysin and aminopeptidase B (Ap-B) (22, 23, 24, 25). NRDc belongs to the M16 family of metalloendopeptidases (26), involved in various prohormone- or proprotein-processing events (27). As observed for somatostatin, dynorphin A, ANF-(1–28), or preproneurotensin-(154–170) (28, 29), the NRDc activity is restricted to the N-terminal side of either dibasic sites (28, 30), such as Arg-Arg, Arg-Lys, Lys-Arg, and Lys-Lys (24, 25), or single basic residue flanked by an aromatic or another specific hydrophobic residue (31, 32). Two isoforms of NRDc have been identified, NRD1 and NRD2; NRD2 differs from NRD1 by an insertion of 68 amino acids (corresponding to a 204-nucleotide insertion). Ap-B, a member of the M1 family of metalloexopeptidases (33), exhibits in vitro a strict selectivity toward N-terminal Arg-X and Lys-X bonds from peptides mimicking processing intermediates, as well as toward several substrates, including Arg0–Leu-enkephalin, Arg0–Met-enkephalin, and Arg–1–Lys0-somatostatin-14 (21). Like NRDc, Ap-B may be involved in propeptide- and proprotein-processing mechanisms. Nonetheless, no relevant physiological substrate of NRDc or Ap-B has yet been definitely identified.
The enzymatic mechanism implicated in the processing of MG from glucagon is still unclear despite attempts to solve this question (11, 34). The aim of our study was: 1) to characterize the MG-generating endopeptidase (MGE) activity; and 2) to precisely localize it in the -pancreatic cells, the sole cells of the organism that contain MG in a stored form. To address this issue, we first used the mouse -pancreatic TC1.6 cells, which have been shown to contain both the substrate (glucagon) and the product (MG) in the same secretory granules, as in native -cells (10), explaining why both glucagon and MG are cosecreted (10). For practical reasons, the rat pituitary GH4C1 cell line was used at various steps of our research, and rat hepatic FaO cells allowed us to compare -cell-containing MGE features with previous results obtained on liver (11).
We report here that MGE is not a PC but a set of two metalloproteases, namely NRDc and Ap-B. The rationale for this conclusion is: 1) the spectrum of sensitivity toward inhibitors clearly shows that a metalloprotease with a thiol group in the catalytic site, as well as an aminopeptidase, are involved in the MGE activity; 2) addition of recombinant NRDc in the medium enhances the activity; 3) recombinant Ap-B is able to trim in vitro N-terminal Arg residue(s) from MG-intermediate products; 4) immunodepletion of either enzyme results in the disappearance of the majority of MGE activity from the culture medium of MGE-secreting cells; 5) NRDc and Ap-B RNAs and proteins are present in the three MGE-secreting cell lines studied; 6) both proteins are found in the mature secretory granules of the -cells, close to their substrate, glucagon, and their product, MG; and 7) NRDc is present specifically in glucagon- and MG-containing fractions from perfused pancreas after physiological stimulation by hypoglycemia.
Materials and Methods
Materials
Inhibitors and cations were purchased from Calbiochem (VWR International, Fontenay-sous-Bois, France), Sigma-Aldrich (Aldrich, Saint Quentin Fallavier, France), Merck (VWR International), and Fluka (Aldrich). MG and Tyr25–glucagon-(19–25) used for RIA were synthesized in our laboratory by a solid-phase procedure, purified by HPLC and subsequent analysis of amino acid composition by mass spectrometry, as previously described (35).
Cell culture
The mouse -cells (TC1.6 cell line) were grown in DMEM containing 1 g/liter glucose, whereas the rat anterior pituitary lactotrophic GH4C1 cells and the rat hepatic FaO cells were grown in DMEM containing 4.5 g/liter glucose (DMEM, Invitrogen, Life Technologies, Cergy Pontoise, France), each supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen,) and 1% antimycotic fungizone (Invitrogen) equilibrated with 5% CO2–95% air at 37 C. Cell culture media were changed every 48 h; and, when reaching 80% of confluence, cells were detached with 2.5% trypsin, 0.5 mM EDTA (Invitrogen).
Enzyme Assays
The MGE activity was obtained from conditioned media of all cell lines after repetitive 2-h incubation periods at 37 C without serum. After collection of the media, two other incubations were performed in the same way, and pooled. The resulting cell-conditioned media were then concentrated approximately 300-fold by centrifugation on Centricon (Amicon, Millipore Corp., Bedford, MA), as previously described (36). Enzymatic preparation was stored at –20 C in 30% glycerol before enzymatic assays. All assays were performed in triplicate, using synthetic mammalian glucagon as substrate (50 pmol, i.e. final concentration of 200 nM), in a final vol of 250 μl in 50 mM Tris acetate/2 mM CaCl2 buffer (pH 7.5). All reaction mixtures were prepared at 4 C, preincubated at 37 C for 15 min with or without different inhibitors, and were incubated for 1 h with glucagon. All reactions were stopped by heating the samples at 100 C for 5 min. The amount of MG produced was measured by RIA.
RIA of MG
As previously described (37, 38), we used mono 125I-Tyr25–glucagon-(19–25) as the tracer, Nle29-glucagon-(19–29) as the standard, and a rabbit anti-MG serum named SAR (37), which specifically recognizes the N-terminal epitope present in MG and masked in glucagon, the mother-molecule (the cross-reaction of glucagon in the assay is about 0.2%). We evaluated the MGE activity by reference to the control assay in which the enzyme was incubated for 1 h at 37 C with glucagon, at pH 7.5. In the presence of different inhibitors, the observed values were computed with reference to the control assay considered as 100%. Results are presented as means ± SEM of at least three experiments.
RT-PCR
Total RNA was extracted from each cell line by the RNAzol method (Extract All, Eurobio, Les Ulis, France). Equal amounts of total RNA (1 μg) from each sample were treated with DNase I (Amplification Grade, Invitrogen) and reverse-transcribed at 42 C using Superscript II Reverse Transcriptase (Invitrogen). To amplify specific regions of Ap-B and NRDc cDNAs, sequence-specific primers were designed on the basis of their nucleotide sequences obtained from GenBank. After an initial denaturation step at 94 C (3 min), thirty cycles of PCR were carried out at 94 C (30 sec), 62 C (30 sec), and 72 C (1 min) in a robocycler gradient 96 (Stratagene Ltd., Cambridge, UK) using Taq polymerase (Roche Diagnostics, Meylan, France). Sense and antisense primer sequences used to amplify rat Ap-B cDNA template (Accession number U61696) were 5'-ACC TTT GTC ACC CCG TGC CTG CTA-3' and 5'-GGG TTT TCC TCT CCA GAC ACA TCC-3', respectively. These specific primers were designed to produce a 233-bp-long DNA fragment. For mouse Ap-B cDNA template (39), sense and antisense primers were 5'-CAA GGA GGA ATA CAG TGG GGT CAT-3' and 5'-CAT TGT AGG TGT CAT CGG GGT CCA-3', respectively. These primers led to the production of a 431-bp-long DNA amplification product.
For rat NRDc cDNA template (Accession number L27124), sense and antisense primers were 5'-GTG GAG GGT AAA ACA GGA AAT GCA-3' and 5'-AGG CAT CAA ATC CAT TCT CAT CTG-3', respectively. The rat 5' primers were chosen 187 bp upstream from the 204 nucleotides insertion (corresponding to the 68 amino acids coding sequence), present only in the NRD2 isoform, to identify which RNAs are present in this cell (40). These primers discriminate between both NRDc isoforms (NRD1 and NRD2), leading to the production of two different fragments of 385 and 589 bp in length, respectively. For mouse NRDc (accession number U86112), primers were 5'-CGT TCT TCT CTC TGG TGC TAA TGA-3' and 5'-ATA GGC GGG TTC GGC AAG GTT GTG-3', respectively, resulting in an amplification product 373 bp long. Amplified RT-PCR products were separated on 2% agarose gel and visualized by ethidium bromide staining.
Western blotting
Total proteins were extracted from cells grown in 10-cm culture dishes with 1 ml lysis buffer (50 mM HEPES, 1% Triton X-100, 1 mM EDTA, 30 mM pyrophosphate, 1 mM vanadate, 1 mM PMSF, 1 mg/ml bacitracin) for 30 min at 4 C. Cell lysates were then centrifuged for 30 min at 4 C at 14,000 rpm. The protein concentration of the collected supernatant was determined by Bradford miniassay. For Western blot analysis, proteins were denatured by boiling during 5 min in Laemmli buffer, separated on a 5% or 7.5% sodium dodecyl sulfate-polyacrylamide gel for the identification of NRDc or Ap-B, respectively, and transferred on a nitrocellulose membrane. After blocking overnight at 4 C in PBS containing 0.1% Tween 20 and 5% BSA or 10% of milk blocking buffer for NRDc and Ap-B, respectively, membranes were probed with rabbit polyclonal anti-NRDc serum (diluted 1:5000) directed against the acidic domain of NRDc (29, 41), or with a rabbit polyclonal anti-Ap-B serum (diluted 1:2000) directed against purified Ap-B (21), in blocking buffer for 1 h at room temperature. The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (diluted 1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA). The antigen-antibody complexes were visualized using the ECL chemiluminescence detection kit (Amersham Life Sciences, Saclay, France) and observed with a Kodak Image Station 2000 system (Eastman Kodak Co., Rochester, NY).
Isolation of islets of Langerhans
Islets were isolated from adult male Wistar rats using collagenase digestion and collected after centrifugation on a Ficoll density gradient as previously described (10). For electron microscopy studies, islets were incubated for 1 h at 37 C in Krebs-Ringer bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4.7H20, 10 mM NaHCO3, pH 7.4) containing 1 g/liter BSA and 2.8 mmol/liter glucose. For immunofluorescence experiments, islets were cultured in RPMI-1640 containing 5.6 mmol/liter glucose, 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/liter glutamine for 24 h.
Immunofluorescence
After dissociation with 0.025% trypsin/0.01% EDTA, islets were seeded on poly-L-lysine-coated-(Sigma-Aldrich) LabTek Chamber Slide System. After a 7-d culture period, they were fixed with 2% paraformaldehyde in PBS for 20 min and permeabilized by a 5-min incubation in 0.1% Triton X-100. After saturation with 2% BSA solution for 20 min, cells were incubated overnight with the same Ap-B or NRDc antiserum used for Western blotting (diluted 1:50) and a sheep antiglucagon antibody (diluted 1:100) directed against centro/N-terminal glucagon (Biogenesis, Poole, UK). After washing, a fluorescein isothiocyanate-conjugated rabbit antisheep (Vector Laboratories, Burlingame, CA) and a Texas Red-conjugated goat antirabbit antibody (Vector Laboratories) were applied separately to the cells for 1 h (both diluted 1:100). Cells were finally mounted in Citifluor (Citifluor LTD, London, UK) and observed with a Bio-Rad (Hercules, CA) confocal microscope equipped with an argon-krypton laser using the facilities of the Centre Régional d’Imagerie Cellulaire (Montpellier, France). The negative control was performed by incubating the cells only with the secondary antibody.
Electron microscopy
Isolated islets were fixed with 2.5% paraformaldehyde/0.1% glutaraldehyde in 100 mmol/liter phosphate buffer (pH 7) for 1 h at room temperature. The islets were then washed in 50 mmol/liter NH4Cl-PBS, postfixed in 1% osmium tetroxide for 2 min, dehydrated in an ascending series of ethanol, and embedded in LR White (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin (60 nm) sections were cut using a Reichert ultramicrotome (Ultracut S, Vienna, Austria) and deposited on gold grids. Sections were saturated with 10% fetal calf serum in PBS and incubated overnight at room temperature with a rabbit antiglucagon, anti-Ap-B, or anti-NRDc serum (diluted 1:100, 1:20, and 1:50, respectively), all produced in our laboratories. After washing, an antirabbit antibody (diluted 1:25) labeled with 10-nm gold particles (British Biocell, Cardiff, UK) was applied to the sections for 1 h. Washed sections were finally stained with 2% uranyl acetate for 20 min and observed with a transmission electron microscope (Hitachi H-7100, Hitachi, Düsseldorf, Germany). The specificity of the immune reaction, for both immunofluorescence and electron microscopy, was tested by incubating the sections only with the secondary antibody.
Reverse-phase HPLC
The concentrated supernatant of GH4C1 cells was incubated for 1 h at 37 C with 20 nmol glucagon as substrate in 50 mM Tris acetate/2 mM CaCl2 buffer (pH 7.5), 100 μM bacitracin (to avoid degradation of the peptide produced), and with 1,10-phenanthrolin (1 mM) or amastatin (500 μM). Control was performed with similar incubation contents without inhibitor. Synthetic peptides corresponding to expected intermediate products containing one (R-MG) or two (RR-MG) arginine extensions and the final product, MG, were run on HPLC to calibrate the column. The incubation medium containing the resulting cleavage products was applied to an Uptisphere silica ODB column (Interchim, Montlu?on, France) at room temperature. The column solvent was water + TFA 0.05% (solvent A) for aqueous phase and acetonitrile (solvent B) for the organic phase. The HPLC system was operated at 1 ml/min and 25% of solvent B. Elution was conducted with a linear gradient of 25–40% of solvent B for 40 min at 1 ml/min. Incubation with purified rat Ap-B, was performed for 1 h at 37 C with 1 nmol of processing intermediates (RR-MG or R-MG) as substrates in 0.1 M borate buffer, containing 150 mM NaCl (pH 7.2). The cleavage products were separated by HPLC using the protocol described above.
Immunoprecipitation procedure
Concentrated supernatants from TC1.6 were incubated with a primary antibody directed against NRDc (diluted 1:200) or Ap-B (diluted 1:500) overnight at 4 C. Immunocomplexes were then precipitated from the supernatant with protein A/G plus-Agarose (Santa Cruz Biotechnology) for 4 h at 4 C. After a 5-min centrifugation at 3000 rpm, the supernatants were collected. Standard reaction assays were performed using 50 pmol glucagon incubated for 2 h at 37 C. Reactions were stopped by boiling at 100 C, and the MG produced was assayed by RIA, as described above. Controls were performed by omitting the primary antibody or by using an unrelated primary antibody used for immunoprecipitation of the PI3-kinase catalytic subunit (Tebu-bio s.a, Le Perray en Yvelines, France).
Isolated perfused rat pancreas fractions
Fractions from rat isolated perfused pancreas obtained as described (10) were concentrated by lyophilization and analyzed for their content in NRDc by Western blotting after immunoprecipitation of the enzyme, both procedures being performed according to the above Western blotting and Immunoprecipitation procedure sections.
Statistical analysis
All results are presented as mean values ± SEM together with the number of individual determinations. Results were expressed as mean ± SEM and data compared using the Student’s t test. Groups of data were considered significant at P < 0.01 (**) or P < 0.05 (*).
Results
From the first attempts to isolate the enzymatic system (MGE) responsible for the secondary processing of glucagon into MG (11, 34, 38), it appeared that the highly purified MGE from rat liver membranes (24, 38) had the features of a metalloprotease with a requirement for a free thiol group in the catalytic site. Because the single cell in the organism that stores and secretes MG is the glucagon-secreting -cell of the islets of Langerhans, we studied the presence of an activity of the MGE type in the conditioned medium of the TC1.6 cell line. The rationale for using this cell line was: 1) these cells are well differentiated with features close to that of the native -cells of the islets of Langerhans; in particular, they are known to store and secrete glucagon and MG (10); and 2) we had direct access, via the culture medium of this cell line, to the secreted products in which we expected to find the enzymatic system producing MG, which was shown to be present in the same secretory granules as glucagon. From that point, we started to characterize the MGE activity present in the conditioned medium of the TC1.6 cell line. We extended our observations to a hepatic cell line (FaO), which represents the equivalent in culture of the hepatic cells in which we had described MGE originally, and to the GH4C1 cell line, an apparently not physiologically relevant cell line of pituitary origin but with an endocrine and highly transformed character (unlike the TC1.6), which makes it a suitable source for running experiments which necessitate large amount of material.
Effects of proteinase inhibitors on the MGE activity released from the TC1.6 cell line
After having established the presence of a MGE activity in the concentrated culture medium of TC1.6 cells, we analyzed its enzymatic features using various proteinase inhibitors. The results were compared with previous data obtained with purified MGE from rat liver plasma membranes (11, 34, 38).
As shown in Table 1, we observed a modest, yet significant, inhibitory action of EDTA, a profound inhibition induced by the metal chelator 1,10-phenantrolin and clear-cut effects of thiol reagents, in particular dithiothreitol (DTT). We conclude that the enzymatic system producing MG from glucagon in -cells shares, with that present in liver, a thiol-dependent metalloendoprotease character, both exhibiting a similar inhibitory profile (11, 38). Moreover, the MGE activity present in hepatic FaO cells shares similar enzymatic features toward EDTA, 1,10-phenantrolin, and DTT (about 25%, 7%, and 20% remaining MGE activity at the highest concentration, respectively; data not shown). In addition, the aminopeptidase inhibitors amastatin and bestatin inhibited the MGE activity at low doses, suggesting the involvement of an aminopeptidase activity in the maturation process present in TC1.6 cells (Table 1), as well as in FaO cells (around 52% and 60% at the highest concentration, respectively; data not shown).
TABLE 1. Comparison of proteinase inhibitors effects on the MGE activity released from the TC1.6 cell line
Effects of zinc, cobalt and calcium ions on MGE activity from TC1.6 cells and its restoration by metallic ions after extinction by 1,10-phenanthrolin
The apparent metalloprotease character of MGE led us to test the effect of zinc, cobalt, and calcium ions on the MGE activity. As shown in Table 2, the addition of zinc or cobalt cations, known to control the catalytic activity of metalloendoproteases, dose-dependently inhibited the MGE activity secreted by the TC1.6 cells; whereas calcium, necessary for a PC activity, had no effect on MG production. This strongly supports the metallopeptidase character of MGE, which is fully consistent with previous data on the MGE activity obtained from hepatic plasma membranes (11, 34, 38), as well as from hepatic FaO cells, similarly affected by zinc and cobalt addition (data not shown).
TABLE 2. Effects of zinc, cobalt and calcium ions on the MGE activity from TC1.6 cells and its cation reactivation profile after metal chelation by 1,10-phenanthrolin
Furthermore, after its suppression by the metal chelator 1,10-phenanthrolin, the MGE activity was restored by addition of zinc or cobalt, with a blunting effect at higher concentrations, with, again, no effect of the calcium ion.
These observations fit well with the known sensitivity of metalloendoproteases toward the metallic ion implicated in the catalytic activity of such enzymes (24), and rules out the implication of a PC, which belong to a family of calcium-dependent serine endoproteases (16, 17). The sensitivity toward the metal ions, both in their suppressing effect and in their restoration capabilities, was very similar for MGE obtained from FaO cells, again demonstrating the presence of the same enzymatic system in both tissues.
Ap-B, potential component of MGE, is able to cleave intermediate products of glucagon processing
In view of the effects of the aminopeptidase inhibitors amastatin and bestatin, we have hypothesized that MGE cleaves glucagon sequentially, including a first cut by a metalloendoprotease at the level of the basic doublet followed by trimming of the basic amino acid(s) by an aminopeptidase of the B type (aminopeptidase-B or Ap-B).
From our studies in various cell lines, it appeared that an abundant MGE activity was present in the conditioned medium of the pituitary GH4C1 cells, with all the enzymatic features found in the media from TC1.6 and FaO cells (data not shown). According to the apparent identity of MGE from the three cell lines and considering the abundance of MGE activity in the supernatant of GH4C1 cells, we have used the supernatant of those cells as the MGE archetype at that step of the work. The concentrated culture medium of GH4C1 cells was incubated with glucagon for 1 h at 37 C, with or without amastatin or 1,10-phenanthrolin at concentrations observed to inhibit MGE activity, and run on HPLC after column calibration using synthetic peptides corresponding to expected intermediate products (R-MG or RR-MG) and the final product, MG. As shown in Table 3A, we observed the presence of the three peptides (RR-MG, R-MG, and MG). Addition of amastatin resulted in the complete disappearance of MG, whereas the amounts of intermediate products, RR-MG and R-MG, increased. On the other hand, the production of RR-MG, R-MG, and MG was completely suppressed by addition of 1,10-phenanthrolin (Table 3A). These data strongly suggest that, in accordance with our previous working hypothesis, glucagon is processed into MG by a metalloendoprotease cleaving the substrate at the level of the Arg17–Arg18 site, followed by an aminopeptidase, which trims the remaining arginine residue(s), leading to the final product, MG.
TABLE 3. Proportion of glucagon intermediate products after incubation of GH4C1 MGE, NRDc or recombinant Ap-B activities with glucagon or other substrates
To further establish the existence of this sequential cleavage, recombinant Ap-B was incubated with its potential substrates RR-MG and R-MG (Fig. 1). This resulted in MG production using R-MG as substrate (Fig. 1B and Table 3B), whereas incubation with RR-MG led to R-MG and MG production (Fig. 1C and Table 3B). Furthermore, addition of amastatin reduced the yield of MG, whatever the Ap-B substrate (Table 3B). It is interesting to note that Ap-B was unable to cleave in vitro the basic doublet of glucagon (Table 3B), indicating the absence of endoproteolytic activity of Ap-B, and thus that a metalloendoprotease is necessary, upstream of the aminopeptidase, in the MGE activity. Addition of a double amount of Ap-B resulted in the complete transformation of R-MG into MG (data not shown).
FIG. 1. HPLC profiles of peptides produced after incubation of recombinant Ap-B with potential intermediate products from NRDc activity during the course of in vitro glucagon to MG processing. A, Control run with enzymatic preparation alone. B, Production of MG from R-MG used as the substrate. C, Production of MG and the intermediate peptide (R-MG) from RR-MG used as the substrate.
These results are in full agreement with the hypothesis that Ap-B is responsible for trimming the basic amino acids after the action of an endoprotease on the Arg-Arg doublet of glucagon.
NRDc, potential component of MGE, is able to process glucagon into MG
Because a primary endoproteolytic mechanism is necessary to release Ap-B substrates and because such a sequential cleavage, involving NRDc/Nardilysin and Ap-B metalloproteases, has already been identified in the processing of somatostatin-28 into somatostatin-14 (22, 23, 24, 25), we have hypothesized that the endoprotease might be NRDc. To address this issue, recombinant NRDc was added to the medium from GH4C1 cells (containing endogenous Ap-B, necessary to uncover the MG epitope for MG RIA), and MG production was evaluated and compared with its production by the MGE from GH4C1 alone.
As shown in Table 3C, addition of recombinant NRDc to the GH4C1 medium significantly increases the MG production, although to a low extent (see Discussion).
Altogether, these data are fully compatible with our hypothesis that MGE is composed of a set of two metalloproteases: NRDc and Ap-B.
The MGE activity is inhibited after immunoprecipitation of NRDc or Ap-B from a crude MGE preparation
In an attempt to confirm our preceding results, we used another technical approach, i.e. immunoprecipitation of either the NRDc or the Ap-B from the MGE-containing conditioned medium from our cell lines by using specific antibodies. The immunodepleted culture medium was incubated with the substrate glucagon, and the remaining activity was measured. As shown in Fig. 2A, a majority of the MGE activity was removed by the immunoprecipitation procedure, with a 85.3% and 85.2% reduction from the GH4C1 cells, using the anti-NRDc and the anti-Ap-B antibodies, respectively.
FIG. 2. Effect of immunodepletion of NRDc or Ap-B on MG production from GH4C1 or TC1.6 MGE. A, After immunoprecipitation of NRDc or Ap-B using the corresponding specific antibody (NRDc or Ap-B) or without antibody (control, noted CTL) from the GH4C1 cells supernatant containing MGE activity, the supernatant was incubated with glucagon as the substrate for 2 h at 37 C. MG production was measured by MG RIA. B, Similar immunoprecipitations were performed using the supernatant from the TC1.6 cells, and the same experiments were carried out. The results represent means ± SEM from at least three separate experiments. * or **, Significantly different from the control (P < 0.05 or P < 0.01, respectively; Student’s t test). C, As a negative control, we used, on the MGE preparation from TC1.6 cells, an unrelated antibody (Ab) against the PI3-kinase catalytic subunit to exclude any spurious effect in the immunoprecipitation experiment.
A similar profile was obtained for the MGE activity from TC1.6 cells (Fig. 2B): the activity was inhibited by 86% or 87.3%, with anti-NDRc and anti-Ap-B antibodies, respectively. As a negative control, we used an unrelated antibody, i.e. a PI3-kinase catalytic subunit-directed antibody, known to be active in separate PI3-kinase immunoprecipitation experiments. No decrease in the MGE activity was noted (Fig. 2C), ruling out any spurious effect of the anti-NDRc or the anti-Ap-B antibody.
These results strongly strengthen precedent data and clearly indicate that both NRDc and Ap-B are deeply involved in the MGE activity, by acting sequentially to release MG from the mother-hormone.
NRDc and Ap-B, the two potential components of MGE, are present in MG-producing tissues
Our results led us to conclude that the MGE activity responsible for the in vitro processing of glucagon into MG is composed of both NRDc and Ap-B. However, neither the expression nor the subcellular localization of NRDc and Ap-B has yet been established in these cell lines. To address this issue, we used four complementary approaches: RT-PCR, Western blotting, immunofluorescence, and electron microscopy.
By RT-PCR, we identified amplified PCR products of the rat and mouse Ap-B at the expected size (Fig. 3, A and B). As described in Materials and Methods, two alternatively spliced isoforms of NRDc could be detected in rat cell lines (rNRD1 and rNRD2; 385 bp and 589 bp amplification bands, respectively) (Fig. 3A), whereas a single amplification band of 373 bp for the mouse NRDc could be detected in the TC1.6 cell line (Fig. 3B). The PCR products were analyzed by sequencing the corresponding gel-purified bands (data not shown). The main isoform of NRDc present in rat cells is NRD1, whereas a faint band corresponding to NRD2 was visible in both rat cell lines (Fig. 3A). In addition, we observed some other RT-PCR bands in the rat FaO cell line (Fig. 3A, left), but the scarcity of the material precluded any further identification.
FIG. 3. Presence of both NRDc and Ap-B RNAs and proteins in GH4C1, TC1.6, and FaO cells. A, Results from PCR after reverse-transcription of RNAs from GH4C1 and FaO cell lines, by using specific primers (see Materials and Methods). Each PCR product was visualized with ethidium bromide. Lanes 1 and 3, Rat Ap-B primers; lanes 2 and 4, rat NRDc primers. The product lengths were 385 bp or 589 bp and 233 bp, respectively for NRD1 or NRD2 and Ap-B-amplified products. B, RT-PCR of mouse TC1.6 cDNAs. Lane 1, Mouse Ap-B primers; lane 2, mouse NRDc primers. The product lengths were 431 bp and 373 bp for Ap-B and NRDc amplified products, respectively. C, Western blot analysis of Ap-B in GH4C1, FaO, and TC1.6 cell lines. Total cell lysates (35 μg) were processed on 7.5% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane and incubated with the specific antibodies against Ap-B. D, Similar protein extracts were separated on a 5% polyacrylamide gel and incubated after transfer with the specific antibodies against NRDc. Partially purified NRDc and Ap-B proteins were used as controls. Standard proteins (molecular mass markers, in kDa) are indicated along the blot.
Using specific antibodies developed against purified Ap-B (21) or against the acidic stretch of NRDc (41), we show the presence of both proteins by Western blotting in the different cell lines. Indeed, the GH4C1 and FaO cell lines contain the 72-kDa Ap-B form (Fig. 3C, lanes 1 and 2, respectively). In TC1.6 cells (Fig. 3C, lane 3), we observed a band of weaker intensity with the same migration profile as the Ap-B standard (Fig. 3C, lane 4) and those from rat cell lines (Fig. 3C, lanes 1 and 2).
In Fig. 3D, we observed the presence of a major band (140 kDa) corresponding to NRD1 (Fig. 3D, lanes 1–3), with a migration profile similar to that of the NRDc control (Fig. 3D, lane 4). We also observed an upper band corresponding to NRD2 (145 kDa), with a weaker intensity, suggesting its lower expression in the rat cell lines, whereas it was undetectable in the TC1.6 cells. These results are in line with the presence in the rat cell lines of the 385 bp as the major amplicon corresponding to NRD1 and the minor 589-bp amplicon corresponding to NRD2 (Fig. 3A).
Hence, our results show the expression of mRNAs coding for both NRDc and Ap-B, as well as their translation products in the glucagon- and MG-producing -cells.
NRDc and Ap-B are colocalized with their substrate glucagon in the -cells
In immunofluorescence confocal microscopy studies (Fig. 4), glucagon staining appears to be granular in -cells freshly isolated from rat islets of Langerhans, the punctuated staining corresponding to glucagon-containing secretory granules (Fig. 4, A and D). The Ap-B staining, also granular, appears to be similar to that observed for glucagon (Fig. 4B).
FIG. 4. Localization of glucagon and Ap-B or NRDc by double immunofluorescence. Dissociated pancreatic islets, cultured for 7 d on LabTek slides, were fixed with 2% paraformaldehyde, followed by immunoreaction. Pancreatic -cells were double-immunostained with antiglucagon (A and D) and anti-Ap-B (B) or anti-NRDc (E) antibodies. Immunofluorescence was analyzed by a dual-channel confocal microscope. Glucagon and Ap-B or NRDc stainings appear as green (A and D) and red (B and E), respectively. Coincidence of both fluorescences results in the appearance of a yellow color (C and F). Pictures G and H represent the double staining of TC1.6 cells with antiglucagon and anti-Ap-B or antiglucagon and anti-NRDc antibodies, respectively. Picture I is a negative control (omission of the primary antibody). Scale bars, 10 μM.
Similar observations were made for NRDc (Fig. 4E) with the antibody used for Western blotting and directed against the acidic stretch domain of NRDc. Coincidence of the two fluorescences shows that both enzymes obviously colocalize (Fig. 4, C and F) with glucagon in normal -cells isolated from rat islets of Langerhans. The staining appeared as discrete, punctuated, which is typical for a granular staining, observed for a secretory vesicle localization. Together with the already known colocalization of glucagon and MG in the granules (10), this observation demonstrates that the substrate (glucagon), the product (MG), and the processing enzymes colocalize in secretory granules and strongly supports our hypothesis.
As shown in Fig. 4, G and H, respectively, a similar Ap-B/glucagon or NRDc/glucagon colocalization was observed in the glucagon-secreting TC1.6 cells. Figure 4I demonstrates the specificity of the labeling that disappeared when the first antibody was omitted.
Even more precise evidences for the colocalization of the substrate glucagon and the processing enzymes were obtained using gold particles immunostaining at the ultrastructural level (Fig. 5). Indeed, both proteins were detected at the periphery of the mature glucagon secretory granules, again in line with the already observed MG at that place (10). These results further demonstrate the presence of Ap-B and NRDc in glucagon secretory vesicles of the -cells, at the periphery of mature glucagon-containing granules. In addition to its presence in secretory granules, NRDc could also be observed at cytosolic locations.
FIG. 5. Subcellular localization of Ap-B and NRDc in pancreatic -cells of rat islets by immunogold electron microscopy. Electron microscopy was performed on ultrathin sections of isolated islets from rat pancreas after immunostaining according to the gold particles technique as described in Materials and Methods. The figures represent part of the cytoplasm of pancreatic -cells, rich in secretory granules. Staining was performed using rabbit antiglucagon (upper panel), rabbit anti-Ap-B (middle) or rabbit anti-NRDc (lower) antibodies, followed by secondary antibody coupled with 10-nm gold particles (directed against rabbit antibodies. Magnification, x80,000.
NRDc, the major component of MGE, is cosecreted with glucagon and MG
To further ascertain that NRDc is present in the secretory pathway, in contrast to what is usually thought, and is secreted together with the substrate glucagon and the product MG, we selected preserved fractions of the experiments that allowed us to demonstrate cosecretion of glucagon and MG (10). As shown in Fig. 6, neither the insulin-containing 11-mM fraction immediately before the switch to 2.8 mM nor the fraction collected 2 min after this switch, which do not contain significant amounts of glucagon or MG (10), show any detectable amount of NRDc, using the immunoprecipitation procedure followed by Western blotting. In contrast, two fractions selected close to the secretion peak of glucagon and MG [7 and 12 min of hypoglycemic stimulation of glucagon and MG secretion (see Ref.10)] clearly contain NRDc (Fig. 6). This proves that, upon physiological stimulation of glucagon and MG secretion by decreasing the glucose concentration, NRDc is cosecreted with both peptides and thus is present in the regulated secretory pathway. As expected, similar results were obtained using an Ap-B antibody, confirming the presence of Ap-B together with NRDc in glucagon- and MG-containing fractions (data not shown).
FIG. 6. Presence of NRDc in glucagon- and MG-containing fractions from perfused rat pancreas after physiological stimulation by hypoglycemia. Selected fractions from isolated perfused rat pancreas (10 ) (see Materials and Methods) were analyzed, after NRDc immunoprecipitation, by Western blotting for their NRDc content. 11 mM, Fraction at the end of the insulin-stimulatory period, which does not contain either glucagon or MG (10 ). The three next fractions correspond to 2, 7, and 12 min after glucagon (and MG) stimulation period by hypoglycemia (2.8 mM glucose). Fractions 7 and 12 were selected close to the secretion peak of glucagon and MG.
Discussion
MGE, responsible for the secondary processing of the mother-hormone glucagon into MG (11, 34, 38), controls the glucagon/MG balance, giving this enzyme a pivotal role in this new level in the regulation of metabolism. In the present report, we characterize the glucagon-processing enzyme contained in MG-producing tissues and demonstrate that MGE is made of two metalloproteases, NRDc and Ap-B, which act sequentially, leading to the release of the final active product, MG. We have also identified their cellular and subcellular localization in the -cells of islets of Langerhans, where both enzymes are colocalized in mature glucagon-containing secretory granules, beside their substrate glucagon, as well as the product, MG, and are secreted together with the two peptides.
Our data yield new insights into prohormone processing, classically initiated in the trans-Golgi network by one or several specific endoproteases of the PC type at monobasic, dibasic, and/or multibasic residues (12, 13, 16, 17). Our data fit with the fact that the secondary processing of glucagon into MG is distal to those of proglucagon, which occurs, in -cells, via action of prohormone convertase-2 (42) and thus should be considered as an additional level of prohormone processing, glucagon being considered, in that case, as a prohormone. The metallic ion sensitivity of MGE (notably for zinc and calcium) is consistent with a metalloendoprotease activity (26) and excludes the involvement of a calcium-dependent serine endoprotease of the PC type (16, 17). A similar cleavage scheme has already been shown for the in vitro processing of somatostatin-28 into somatostatin-14, involving NRDc and Ap-B (22, 23, 24, 25). Concerning this question, the evidence that no somatostatin-14 was observed in pancreatic -cells or in brain from PC2-null mice (43, 44) does not rule out the implication of the NRDc and Ap-B system in some tissues, because there is evidence that prosomatostatin processing is not uniform (45) and that prosomatostatin processing up to somatostatin-14 necessitates, in some tissues, a somatostatin-28 step which appears to be PC2 dependent (43, 44, 45).
The involvement of Ap-B in prohormone processing has already been shown to occur either in the secretory pathway, at the plasma membrane, or at both localizations (46). This ubiquitous enzyme, present in endocrine and nonendocrine cells (22, 47) and associated with secretory vesicle membranes from cattle pituitary (46), neurons, and PC12 (47), was described to be secreted both via the constitutive and the regulated pathways (21, 33, 47).
Besides, NRDc localization is more puzzling. The enzyme has been found mainly localized in cytoplasm with significant amounts associated with microtubular structures and secreted by some cell types (48). NRDc activity, present in culture media, has also been found in other intracellular compartments among which secretory granules (49). Although controversial, NRDc was supposed, as shown for PCs, to mature prohormones at dibasic sites within the secretory pathway in cytoplasm and/or at the cell surface (50, 51). This assumption, despite the lack of definite function for the enzyme, was reinforced by the presence of two putative N-glycosylation sites together with a modest putative signal peptide. Besides, a Met49 NRDc isoform, translated from the second initiation codon, has been observed in rat testis (52); the lack of signal peptide in that isoform excluded a priori its potential involvement in prohormones processing in secretory granules of endocrine cells (41, 52). These discrepancies may be explained by a tissue-specific distribution of NRDc isoforms, with a possible expression of Met1 NRDc (containing the signal peptide) in pancreatic -cells and not in other endocrine cells. Because our immunohistochemical experiments were performed using an antiacidic domain of NRDc antibody, we could not discriminate between Met1 and Met49 NRDc isoforms. Whatever the molecular explanation, our data using both morphological and biochemical approaches demonstrate that both enzymes are present in the regulated secretory pathway, at least in -cells.
Interestingly, different molecular forms of cathepsin L have recently been implicated in the production of (Met)enkephalin from its precursor, and its presence demonstrated in secretory vesicles (53). Similarly to the predicted cleavage specificity of MGE, the cathepsin L isoform present in secretory vesicles requires the presence in the intermediate products of N-terminal basic extensions later trimmed by an Ap-B-like activity (53, 54). The present data are thus compatible with the hypothesis that the isoform present in -cells differs from that present in rat testis, or that a chaperone protein retains Met49 NRDc at the periphery of secretory granules. On the other hand, glucagon is as a rather poor substrate for NRDc in vitro (Prat, A., unpublished, Table 3C), when compared with other known substrates such as dynorphin A or somatostatin (28, 29). This might, at a first sight, appear as incompatible with a physiological relevance of the proposed mechanism. This apparently poor adequacy between the enzyme and the substrate is certainly meaningful in this particular case, because only a marginal portion (3–5%) of glucagon is transformed in vivo into MG in rat -cells (10), as well as in humans (55) and at a similar rate for circulating glucagon in target tissues, thanks to the presence of MGE at the cell surface (8, 11, 34, 38). This partial processing is, on the contrary, particularly relevant from a physiological point of view (10), because it fits with the fact that MG (active in the subpicomolar range) is at least three orders of magnitude more potent than glucagon (active in the nanomolar range) on target tissues. In addition, it should be considered that a very efficient processing of glucagon into MG, by antagonizing totally the metabolic effects of the mother-hormone would be antinomic, regarding the counter-regulatory hormone status of glucagon. A recent study (56) has described the production of a small amount of MG among 13 different degradation products, probably by a 170-kDa serine protease; this process observed in hepatic endosomes corresponds to the degradation of internalized glucagon and differs entirely from the specific processing mechanism described here.
MG generated by the MGE activity is now recognized as an original peptide involved in novel regulatory mechanisms. Indeed, in contrast to its mother-hormone, glucagon, it inhibits locally insulin release within the islets of Langerhans (7, 10), whereas its production from circulating glucagon in target tissues (8, 9, 11) allows a new level of regulation, again in a direction opposite to that of the mother-hormone. Moreover, MG has been recently shown to reduce insulinemia in vivo, whereas it favors the peripheral action of insulin (57). This confers to the small peptide the status of a new entity in the regulation of metabolism. Thus, the ability of MGE to produce from glucagon a factor which negatively modulates pancreatic and peripheral effects of the mother-hormone is, from a physiological point of view, of great interest. Accordingly, the glucagon/MG balance, at the origin of which is MGE, is a new parameter of potential importance to fully understand the molecular and cellular bases of fuel homeostasis and their impairment in metabolic diseases.
The present report provides an essential clue that was missing for a full understanding of the physiological pathways in which the glucagon/MG balance is involved. The two components of MGE being now identified, it will be possible by modulating their expression in vitro (small interfering RNA, overexpression) or in vivo (knock-out, knock-in) to analyze the importance of MG production in the biological regulation as well as to obtain information on its possible role in pathological states, such as type 2 diabetes.
Acknowledgments
We are grateful to Christine Fahy (Centre National de la Recherche Scientifique Unité Mixte de Recherche 7631, Université Pierre et Marie Curie, 75006 Paris, France) for technical assistance in HPLC procedures and Antoine Disset (Centre National de la Recherche Scientifique Unité Propre de Recherche 1142, Montpellier, France) for sequencing the purified PCR products.
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