©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Processing of Mouse Proglucagon by Recombinant Prohormone Convertase 1 and Immunopurified Prohormone Convertase 2 in Vitro(*)

Mark E. Rothenberg (1), Carmen D. Eilertson (1), Kathy Klein (1), Yi Zhou (2), Iris Lindberg (2), John K. McDonald (1), Robert B. Mackin (3), Bryan D. Noe (1)(§)

From the (1) Department of Anatomy and Cell Biology, Emory University, Atlanta, Georgia 30322, the (2) Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, and the (3) Department of Biomedical Sciences, Creighton University, Omaha, Nebraska 68178

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mouse tumor cell line TC1-6 was used as a model system to examine the post-translational processing of proglucagon. Determination of the mouse preproglucagon cDNA sequence and comparison with the published sequences of rat and human preproglucagons revealed nucleic acid homologies of 89.1 and 84%, respectively, and amino acid homologies of 94 and 89.4%, respectively. Immunohistochemical analyses with antibodies directed against PC2 and glucagon colocalized both the enzyme and substrate within the same secretory granules. PC1 was also immunolocalized in secretory granules. Cells were metabolically labeled with [H]tryptophan, and extracts were analyzed by reverse-phase high pressure liquid chromatography. Radioactive peptides with retention times identical to those of synthetic peptide standards were recovered and subjected to peptide mapping to verify their identities. To determine the potential role of PC1 and PC2 in proglucagon processing, H-labeled proglucagon was incubated in vitro with recombinant PC1 and/or immunopurified PC2. Both enzymes cleaved proglucagon to yield the major proglucagon fragment, glicentin, and oxyntomodulin, whereas only PC1 released glucagon-like peptide-I from the major proglucagon fragment. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. These results suggest a potential role for PC1 and/or PC2 in cleaving several of the normal products, excluding glucagon, from the mouse proglucagon precursor.


INTRODUCTION

Many biologically active peptides and neuropeptides are initially synthesized as larger precursors that are cleaved at monobasic, dibasic, or tetrabasic pairs of amino acids by specific endoproteases to release the biologically active product(s) (1, 2) . The existence of mammalian endoproteases with the selectivity to process at specific sites within a precursor has been known for some time. Their physiological importance is exemplified by the broad spectrum of precursors they process, which includes peptide hormones, growth factors, receptors, and envelope glycoproteins of viral pathogens (3, 4, 5, 6) . The name that is most often used for the endopeptidases that are specific for cleavage at dibasic amino acids ( e.g. Lys-Arg) is prohormone/proprotein convertases (PCs)()(7, 8, 9, 10) . PCs have also been shown to process at single arginine sites or at sites having more than two basic amino acids (7, 9, 11) .

PCs were found to have characteristics similar to those of the family of subtilisin-like proteases related to the yeast kex2 gene product (4, 11) . Several mammalian and nonmammalian homologues of kex2 have been characterized. Furin, the first to be identified, recognizes the tetrabasic cleavage motif Arg- X-Lys/Arg-Arg (10, 12, 13, 14, 15) . Several other prohormone-processing enzymes have also been cloned and sequenced, including PC1 (also referred to as SPC3) (16, 17, 18) , PC2 (16, 17, 18, 19) , the paired basic amino acid-cleaving enzyme (PACE) family PCs (13, 20) , PC4 (21, 22) , PC5/6 (PC6A) and PC6B (23, 24) , and PC7 (25) . PC1 and PC2 are expressed predominantly within endocrine and neuroendocrine cells and tissues (17, 19, 23, 26, 27) , while furin (15, 28) , PACE4 (13) , and PC6 isoforms (23, 24) are expressed ubiquitously. PC6A (23, 29) has been localized only within a subset of endocrine and nonendocrine cells ( e.g. pancreatic islets and gut endocrine cells), while PC4 (21, 22) is expressed primarily within testicular germ cells. To determine if the PCs were indeed authentic prohormone convertases, cotransfection, antisense, and in vitro experiments were performed (3, 30, 31, 32, 33) . The combined results from these studies and others imply that PCs have a direct functional role in the proteolytic processing of prohormones at either dibasic or monobasic cleavage sites.

We have initiated studies on the processing of mouse proglucagon, an 18-kDa precursor containing glucagon and two glucagon-like peptides (GLP-I and GLP-II). This prohormone is expressed in pancreatic islets, intestine, and brain (34, 35, 36, 37) , and its processing differs depending upon the site of expression. In pancreatic -cells, proglucagon is predominantly processed to glucagon and the major proglucagon fragment (MPGF) (Fig. 1) (36, 37, 38) . To examine the physiological mediators of proglucagon processing in -cells, we sought a cell line that expressed not only the precursor, but also one or more of the PCs. The TC1-6 cell line evolved from a glucagonoma developed in transgenic mice expressing a hybrid gene consisting of a glucagon promoter sequence fused to the SV40 T-antigen oncoprotein (39) . The TC1-6 line was shown to maintain many of its differentiated pancreatic -cell characteristics for >40 passages and to express large quantities of proglucagon mRNA (39) . These cells have also been shown, by radioimmunoassay (RIA), to synthesize large quantities of glucagon, moderate levels of GLP-I, and small amounts of unprocessed proglucagon (39) . Several laboratories have demonstrated that this cell line also expresses PC1, PC2, and PC6A, with PC2 being the most abundant (40, 41, 42) . The primary purpose of this study was to assess the potential roles of PC1 and/or PC2 as physiological mediators of proglucagon processing using an in vitro assay system. A search of the literature and the GenBankData Bank revealed that the primary structure of mouse preproglucagon has not been published. Therefore, we have amplified and sequenced cDNA corresponding to preproglucagon from the mouse-derived TC1-6 cell line. Availability of sequence information allowed us to accurately perform peptide mapping of the metabolic cleavage products of mouse proglucagon. In addition, to support the underlying rationale for the processing studies, we have immunohistochemically colocalized PC2 and glucagon in the same secretory granules of TC1-6 cells.


Figure 1: Schematic representation of proglucagon processing in pancreatic islets. Shaded and hatched boxes indicate potential cleavage products of the precursor. Potential cleavage sites containing a pair of basic amino acids are indicated by black boxes; one basic pair not cleaved in proglucagon synthesizing cells is shown ( RR). The predominant product of processing in pancreatic -cells is glucagon.




EXPERIMENTAL PROCEDURES

Cell Culture

The TC1-6 cell line was obtained from Dr. D. Hanahan (University of California at San Francisco) and was grown in Dulbecco's modified Eagle's medium containing 4.5 g/liter D-glucose and supplemented with 20 mML-glutamine, 100 µg/ml penicillin/streptomycin (Life Technologies, Inc.), and 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Inc., Norcross, GA).

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Poly(A)RNA was isolated and purified from TC1-6 cells using the Mini RiboSep kit (Collaborative Research Inc., Lexington, MA). Approximately 5 µg of mRNA were used per RT-PCR following the protocol described (43) . Oligonucleotide primers were designed to amplify the coding regions of rat preproglucagon and nonhomologous regions of PC1 and PC2. All primers were synthesized on an Applied Biosystems oligonucleotide synthesizer: preproglucagon, 5`-AGCTTCAGTCCCACAAGGCAGAAT (sense) and 5`-GGCCGCAGGAGATGTTGTGAAGAT (antisense); mouse PC1, 5`-AGTGGTGATTACACAGACCA (sense) and 5`-TCCCTTTCAGCCAACAGTAC (antisense); and mouse PC2, 5`-AGGAAT/CCCCGAGGCCGGTGTGGC (sense) and 5`-CTGCCG/CACAAAT/CCCCAGCTC (antisense) (the slashes indicate points of dishomology between known mammalian PC sequences). PCRs were run on an Omnigene Temperature Cycler (National Labnet Co., Woodbridge, NJ) with a program consisting of 30 cycles composed of successive 60-s denaturation periods followed by 2-min annealing periods at 55 °C and then a 3-min elongation period at 72 °C. After PCR, the products were electrophoresed on a 1% low melting agarose gel (SeaPlaque or Nusieve, FMC Corp. BioProducts, Rockland, ME), and bands corresponding to the correct size (detected by ethidium bromide staining) were ligated into the pCRII vector of the TA cloning kit (Invitrogen, San Diego, CA) and transformed into INVF` bacterial cells. Several white bacterial colonies from each transformation were chosen for restriction digests and subsequent DNA sequencing utilizing T7 DNA polymerase (Sequenase version 2.0, Amersham Corp.). At least four clones were sequenced from each transformation in both orientations. The sequence of the plasmid was then matched with the known published sequence of the PCs using MacVector DNA sequence analysis software (Kodak Scientific Imaging Systems, New Haven, CT). In the case of mouse preproglucagon, the sequence was compared with the published rat and human sequences.

Northern Blot Analysis

Total RNA (from 1.5 10cells) was extracted by the method of Chomczynski and Sacchi (44) . Approximately 10 µg of total RNA were denatured with formaldehyde and formamide and electrophoresed at 5 V/cm through a 0.66 M formaldehyde, 1% agarose gel (FMC Corp. BioProducts). An RNA ladder with sizes ranging from 0.25 to 9.5 kb (Life Technologies, Inc.) was also run in a lane adjacent to the RNA samples. Once the samples had migrated the appropriate distance, the gel was subjected to capillary blotting onto a Hybond-N membrane (Amersham Corp.), after which the membranes were baked for 2 h at 80 °C. Approximately 25 µg of mouse PC1, PC2, preproglucagon, and -actin cDNAs were then labeled with 5`-[-P]dCTP (3000 Ci/mmol; Amersham Corp.) using the Megaprime labeling kit (Amersham Corp.). The membranes were prehybridized for at least 1 h at 65 °C in Rapid Hybridization buffer (Amersham Corp.), and the labeled probes (2-3 10cpm/ml) were added directly to the hybridization buffer and hybridized for 2 h at 65 °C. The membranes were washed twice with 2 SSPE (SSPE = sodium chloride, sodium phosphate, EDTA), 0.1% SDS for 10 min at room temperature; once with 1 SSPE, 0.1% SDS for 15 min at 65 °C; and twice with 0.7 SSPE, 0.1% SDS for 15 min at 65 °C and autoradiographed using Amersham Hyperfilm MP at 80 °C for an appropriate length of time.

Antigen and Antibody Production

The following primers were synthesized: human PC1, GATGGATCCAGGGACTCAGCTCTAAATCTC (sense) and CTGGAATTCGCCCGTAATGCCTTTTTGCCA (antisense); and human PC2, GATGGATCCAGAGACATCAATGAGATCGAC (sense) and CTGGAATTCTTTCCCTGTGTATCCCAGCTC (antisense). The human PC1 primers were designed to amplify the region at nucleotides 547-681, while the human PC2 primers were designed to amplify region 421-564. Both sets of primers contained 5`- BamHI and 3`- EcoRI sites for ligation into the multiple cloning site contained in the prokaryotic expression vector pGEX-2T (Pharmacia Biotech Inc.). RT-PCR, as described above, was used to amplify both PCs from mRNA isolated from the human medullary thyroid carcinoma cell line (from Dr. H. Tamir, Columbia University, New York). The fusion protein was expressed and purified as described previously by Smith and Johnson (45) and Lin and Cheng (46) with minor modifications. The pGEX-2T vector without insert was also expressed in order to obtain purified glutathione S-transferase (GST) linked to the thrombin cleavage site for control purposes. The fusion peptides (GST-PC1 or GST-PC2) were then sent to HRP Inc. (Denver, PA) for polyclonal antibody production.

Antibody Purification

Two rabbits (New Zealand White) were used per antigen, with EM4 and EM5 corresponding to GST-PC1 and EM6 and EM7 to GST-PC2. The GST-directed antibodies were separated from whole sera using affinity columns containing GST bound to Affi-Gel 10 resin (Bio-Rad). The GST affinity columns consisted of two 4-ml layers; layer A contained unpurified GST (GST protein and other assorted bacterial proteins), and layer B consisted of purified GST. One-ml aliquots of each serum were loaded onto individual columns at 4 °C and eluted using 15 ml of 10 mM Tris, pH 8.0. The 15 ml of eluate were desalted over a Sephadex G-25 column (Pharmacia Biotech Inc.), lyophilized, and then resuspended in 5 ml of water. The initial titer and specificity of the sera were determined by Western blot analysis using the chemiluminescence Renaissance kit (DuPont NEN) and by RIA.

PC1 and PC2 RIA Development

The RIAs for both PC1 and PC2 were developed using synthetic peptide fragments (Emory University Microchemical Facility) of the original antigen used for immunization. The peptides synthesized corresponded to residues 114-136 for hPC1 (designated hPC1-N23) and 112-136 for hPC2 (designated hPC2-N25). Norleucine was substituted for methionine in each peptide. The hPC1 and hPC2 fragments were iodinated using a chloramine-T procedure as described previously (47) , except that 1 mCi of NaI (>400 mCi/ml; ICN, Costa Mesa, CA) was used, and the reaction was terminated after 1 min. The labeled material was then run over an Econo-Pac 10DG desalting column (Bio-Rad), and aliquots from 200-µl fractions were counted in a -counter. The void volume peak tube plus one fraction on either side were pooled, diluted in assay diluent (9 mM EDTA (Fisher), 0.3% Fraction V bovine serum albumin, and 0.01% sodium azide (Sigma) in 0.05 M phosphate buffer, pH 7.4), and stored at 4 °C. The total volume of each assay was 400 µl, with 200 µl of standard or unknown, 100 µl of antibody, and 100 µl of trace (10,000 cpm/100 µl), all diluted in assay diluent and added on the same day. Final antibody dilutions were 1:8000 for PC1 and 1:12,000 for PC2, with each giving 25% binding in the absence of unlabeled ligand. The standard concentrations ranged from 10 to 1280 pg for PC1 and from 5 to 1500 pg for PC2. After incubation at 4 °C for 48 h, the unbound antigen was precipitated by the addition of 200 µl of assay diluent containing 0.25% dextran (Sigma) and 1% charcoal (Mallinckrodt Speciality Chemicals), incubation for 20 min at 4 °C, and then centrifugation at 3600 rpm at 4 °C for 30 min in a Sorvall RT6000 centrifuge (DuPont NEN). The supernatant was carefully aspirated, and the pellets were counted for 1 min in the -counter using LKB-Wallac software to analyze the standard curve by log-logit transformation.

Immunocytochemical Localization of Glucagon, PC1, and PC2

Cultures of TC1-6 cells were grown on sterile 12-mm glass coverslips (Fisher) to 85% confluency in 24-well plates (Falcon) under the cell culture conditions described above. Prior to fixation, the cells were rinsed twice with Hanks' balanced salt solution and fixed with 4% paraformaldehyde, 0.1 ML-lysine hydrochloride, and 0.01 M sodium periodate (48) at 4 °C for 10 min. After two washes with phosphate/Triton/azide buffer (PTA buffer; 50 mM phosphate buffer containing 0.3% Triton X-100 and 0.1% sodium azide, pH 7.4), 200 µl of 10% normal mouse serum (Sigma) in PTA buffer were added to each well and incubated for 45 min at room temperature. Following a brief rinse with PTA buffer, cells were pretreated with an avidin/biotin blocking kit (Vector Labs, Inc., Burlingame, CA) to minimize nonspecific avidin binding. After subsequent washes, primary antisera to PC1, PC2, or porcine glucagon were added for 1 h at 37 °C. The antiserum was removed, coverslips were rinsed twice with PTA buffer, and 200 µl of the appropriate secondary antiserum were added. After a 1-h incubation at room temperature in darkness, the cells were rinsed with PTA buffer. Coverslips designated for glucagon immunostaining were rinsed, removed, and air-dried before mounting on glass slides. Cells to be stained for PC1 or PC2 received 200 µl of an avidin D-Texas Red or avidin D-FITC conjugate, respectively, at a concentration of 20 µg/ml (Vector Labs, Inc.) and were incubated for 1 h at room temperature in darkness, followed by two rinses in PTA buffer, and air-dried before mounting on glass slides with Entellan (Merck, Darmstadt, Germany). Cells were observed and photographed using a Leitz Laborlux epiillumination microscope equipped with a narrow band-pass filter and housing both rhodamine and FITC cubes.

Primary antisera used to detect PC1 (EM4, GST-purified) and PC2 (EM7, GST-purified) and the antiserum to porcine glucagon produced in guinea pig (Linco Research, St. Charles, MO) were diluted 1:50 (EM4 and EM7) and 1:200 (anti-glucagon). Indirect immunoidentification of PC1 and PC2 was accomplished utilizing a biotinylated mouse anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at a concentration of 1:500. Glucagon immunoreactivity was observed using a Texas Red-conjugated goat anti-guinea pig secondary antibody (Vector Labs, Inc.) at a concentration of 20 µg/ml. All antisera were diluted in PTA buffer.

The specificity of immunostaining was verified by 1) omission of either primary or secondary antisera; 2) parallel incubations with antisera preabsorbed with the appropriate antigen (10 µM porcine glucagon or 30 µM purified PC1/PC2 construct produced in the pGEX expression vector) for 2 h at 37 °C prior to the immunostaining procedure; and 3) using a nonimmune serum control. Cells exposed only to 200 µl of avidin D-FITC or to 20 µg/ml Texas Red reagent demonstrated negligible nonspecific background staining. Rat pituitary tissue, which is known to express PC1 and PC2 (26) , was used as a positive control for PC1 and PC2 immunostaining.

Colocalization of PC2 with Glucagon

The possibility that glucagon and PC2 are colocalized within the same secretory granule was investigated using identical final dilutions of the reagents indicated above. Since both primary and secondary antisera used for PC and glucagon staining were raised in different species, minimal interference was anticipated; however, appropriate control experiments were performed with omission of primary or secondary antisera. Due to the lack of species cross-reactivity and the fact that no differences were observed in sequential staining experiments, primary antisera to PC2 and glucagon, and later the secondary antisera, were added concurrently to the appropriate samples. All coverslips were blocked prior to staining with 10% normal mouse serum in PTA buffer. Cells were observed as described above.

Metabolic Labeling of Mouse Proglucagon

TC1-6 cells were cultured in Dulbecco's modified Eagle's medium in 75-cmflasks (Falcon) until reaching 80% confluency at 2 10cells. Cells were incubated for 30 min at 37 °C in HEPES, pH 7.2, after which they were incubated for 90 min at 25 °C with 250 µCi of [H]tryptophan (20-30 Ci/mmol; Amersham) in HEPES. The cells were extracted at the end of the incubation in 2 ml of 2 M acetic acid and freeze-thawed five times using dry ice/acetone and a 37 °C water bath. The extracts were centrifuged at 10,000 rpm for 10 min, and the pellet was re-extracted with an additional 1 ml of 2 M acetic acid. Supernatants were combined and mixed 1:1 with 30% of a 0.1% trifluoroacetic acid (Pierce), 80% HPLC-grade acetonitrile (Fisher) solution for Sep-Pak Ccartridge (Millipore Corp., Milford, MA) purification. The peptides were eluted from the Sep-Pak column with a solution of 0.1% trifluoroacetic acid, 48% acetonitrile. The purified extract was dried using a Speed-Vac concentrator (Savant Instruments, Inc., Farmingdale, NY) and resuspended in 300 µl of 2 M acetic acid for reverse-phase HPLC (RP-HPLC) analysis.

HPLC Analysis

To separate both synthetic peptides and peptides from cell extracts, we used RP-HPLC protocols similar to those previously described (49) , with some modifications. Separations were performed on a 0.45 25-cm Vydac Ccolumn (5-µm particles, 300-Å pore size). Either 0.5- or 1-min fractions were collected in siliconized glass test tubes, and 0.1 volume aliquots of the radiolabeled fractions were counted in a scintillation counter. For some experiments, 2-3 µg of the following standards were added to the samples to determine the elution position for each: human glucagon (Peninsula Laboratories, Inc., Belmont, CA), hGLP-I- (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) -amide or hGLP-I- (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) -amide, hGLP-II, or oxyntomodulin (all from Sigma). The retention times of the standards were determined by UV absorbance at 210 nm. With the elution protocol employed, we were not able to resolve the two different GLP-I standards; thus, we used the hGLP-I- (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) -amide standard for the remainder of the study. From the HPLC eluates of cell extracts, the fractions corresponding to the retention times of the standards were pooled and evaporated to dryness. The pools were resuspended either in 200 µl of 0.05 M Tris, pH 8.8, for peptide mapping or in 0.5 M sodium acetate buffer, ph 5.3 for in vitro incubations.

Peptide Mapping

Protein content in samples was estimated based on absorbance at 210 nm. Resuspended samples, including precursor and product peaks, were incubated with diphenylcarbamyl chloride-treated trypsin (Sigma) diluted in 0.05 M Tris, pH 8.8, at a ratio of 1:50 (enzyme to substrate) for 2 h at 37 °C. A fresh aliquot of trypsin was then added, and the samples were incubated for an additional 2 h at 37 °C. The final ratio of trypsin to substrate was 1:25. After the 4-h incubation, a 1:25 (enzyme/substrate) ratio of carboxypeptidase B (CPB) (treated with diisopropyl fluorophosphate; Boehringer Mannheim) in 10 µl of 0.05 M Tris, pH 8.8, was added, and the samples were incubated overnight at room temperature. To terminate the reaction, 150 µl of 2 M acetic acid were added, and the tryptic peptides were separated by RP-HPLC. Bovine/porcine glucagon (Sigma), hGLP-I- (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) -amide, and hGLP-II were also subjected to trypsin and CPB treatment, and the HPLC retention times of the resulting peptide fragments were mapped. The samples were eluted with a gradient that was designed to separate tryptic fragments having retention times with differences of <1 min. To optimize resolution, 0.5-min fractions were collected in scintillation vials, 3.6 ml of Ecolite scintillation fluid (ICN) were added, and samples were counted on the scintillation counter. To confirm both the elution profile of mouse glucagon and results from the peptide mapping studies, aliquots from HPLC eluates of unlabeled TC1-6 extracts were assayed in a C-terminally directed glucagon RIA using the O4A antibody (from Dr. R. Unger, University of Texas, Southwestern Medical Center, Dallas).

In Vitro Cleavage of Proglucagon with Recombinant PC1 and Immunopurified PC2

Recombinant PC1 and immunopurified PC2 were prepared as described previously by Lindberg and co-workers (33, 50) . HPLC-purified [H]tryptophan-labeled proglucagon and MPGF pools were each divided equally into four separate 1.5-ml microcentrifuge tubes containing 5 µg of bovine serum albumin (Sigma), vacuum-evaporated, and resuspended in 10 µl of 0.5 M sodium acetate buffer, pH 5.3. The PC2 and PC1 plus PC2 incubations also contained 5 µl of a 10 concentrated inhibitor mixture (10 µM pepstatin, 1 µM E-64, and 1 mML-1-tosylamido-2-phenylethyl chloromethyl ketone) (Sigma). Five microliters of 50 mM CaCland a sufficient amount of water were added to adjust the final volume of the reaction mixture to 50 µl. All aliquots of PC1, whether incubated alone or in conjunction with PC2, were preincubated at 37 °C for 1 h in activation buffer before adding to the sample (32, 33, 50) . All reactions were performed at 37 °C for 15 h, after which 0.2 µg of CPB in 10 µl of 50 mM Tris, pH 8.0, were added to each sample (except the controls) and incubated for 2.5 h at 37 °C. To determine if there was any inherent endopeptidase activity in the CPB, control incubations consisting of proglucagon or MPGF plus CPB were performed in separate experiments. The reactions were terminated with 150 µl of 2 M acetic acid and subjected to RP-HPLC. Cleavage of the substrate Pyr-Arg-Thr-Lys-Arg-MCA (Peptides International Inc., Louisville, KY) was used as a positive control to monitor PC activity for all preparations of PC1 and PC2 employed. Substrate cleavage was assayed on a series 8000C fluorometer (SLM-AMINCO, Urbana, IL) with the excitation and emission wavelengths set at 380 and 460 nm, respectively. The assays were repeated with two different lots of recombinant PC1 and PC2 and with different preparations of all three proglucagon peptides. Data presented in the figures are representative results from at least four different experiments.


RESULTS

RT-PCR Amplification and Sequencing of Mouse Preproglucagon

The RT-PCR procedure used to clone mouse preproglucagon yielded a product of 668 base pairs in length that included the entire coding region of the preproglucagon polypeptide (Fig. 2 A). Sequencing of the mouse preproglucagon cDNA revealed nucleic acid homologies between mouse and either rat or human preproglucagons of 89.1 and 84%, respectively; amino acid homologies were 94 and 89.4%, respectively. There are 10 amino acid substitutions within the N terminus of the mouse preproglucagon precursor when compared with the equivalent rat precursor (Fig. 2 B); glucagon and GLP-I showed 100% amino acid homology among mouse, rat, and human forms. Mouse GLP-II differs by a single amino acid from rat and human GLP-II (serine is substituted for asparagine at position 166), and it also differs from the human sequence by substitution of threonine for alanine at position 174.


Figure 2: Alignment of human, rat, and mouse preproglucagon sequences. A, cDNA sequence comparison. Start and stop codons are indicated by solid black boxes. Regions of complete homology among the three species are boxed. B, amino acid sequence comparison. Boxed areas indicate complete homology. Horizontal bars indicate potential natural cleavage products; tryptophan residues in proglucagon are in boldface. The peptide mapping fragments of glucagon, GLP-I, and GLP-II are designated by dashed lines. These sequence data are available from EMBL/GenBank/DDBJ under accession number Z46845.



The mouse PC1 primers amplified a 550-base pair product, and the mouse PC2 primers amplified a 600-base pair product from TC1-6 mRNA by RT-PCR. The amplified products were sequenced for verification and used as probes, along with the mouse preproglucagon insert, for Northern blot analyses (Fig. 3). The preproglucagon probe yielded an intense signal of 2.4 kb in size. The PC2 probe hybridized to a band of 2.8-3 kb. Only after 3-4 days of exposure was the PC1 signal observed, revealing a band with an approximate size of 4 kb.


Figure 3: Northern blots of TC1-6 total RNA using mouse-derived [P]dCTP-labeled probes. The blots were exposed for the indicated time periods at 80 °C. A: lane 1, mouse -actin, 14 h; lane 2, preproglucagon, 14 h; lane 3, mPC2, 14 h. B: lane 1, mPC1, 3 days. The RNA ladder (0.24-9.5 kb) was run in conjunction with the total RNA.



Antisera and RIA Characterization

Both affinity-purified and unpurified PC1 (EM4 and EM5) and PC2 (EM6 and EM7) antisera were examined by Western analysis and RIA to determine specificity. Both PC1 antisera showed cross-reactivity by Western analysis with the construct GST-PC1, but not with GST-PC2, while both PC2 antisera recognized the GST-PC2 construct, but not GST-PC1 (data not shown). RIAs were performed to further assess the specificity of the PC1 and PC2 antibodies. Cross-reactivities of the PC constructs and various synthetic peptides in the PC1 and PC2 RIAs were determined. The PC1 RIA, having a sensitivity of 10 pg, exhibited 0.0002% cross-reactivity with PC2-N25 and 0.0001% with GST-PC2 at concentrations up to 10 µg. The PC2 RIA, having a sensitivity of 5 pg, showed 0.08 and 0.02% cross-reactivity with PC1-N23 and GST-PC1, respectively, at concentrations up to 10 µg. Neither the PC1 nor PC2 RIA exhibited cross-reactivity with GST. All other synthetic peptides (16 different islet, brain, and gut derivatives) examined in the PC1 and PC2 RIAs exhibited <0.003 and 0.007% cross-reactivity, respectively.

Immunolocalization

Nearly all of the TC1-6 cells displayed intense immunostaining with the glucagon antiserum (Fig. 4 A). Punctate staining, having a size and distribution pattern characteristic of secretory granules, was observed within the cytoplasm. Interestingly, small populations of cells displayed particularly intense staining for glucagon. Incubations with nonimmune serum, antigen-preabsorbed antiserum, and omission of primary or secondary antisera eliminated all staining of this type.


Figure 4: Immunocytochemical detection of glucagon ( A) and PC2 ( B) in TC1-6 cells was detected in single-labeling experiments. Bars, 25 µm. Immunocytochemical colocalization of glucagon ( C) and PC2 ( D) within the same granules (indicated by the arrows) was demonstrated in a separate double-staining study. Bars, 10 µm.



TC1-6 cells displayed both PC2- and PC1-like immunoreactivity, which was also localized within numerous punctate cytoplasmic granules (Figs. 4 B and 5, respectively). Punctate staining was abolished when primary or secondary antisera were omitted and when staining was performed with antisera preabsorbed with the GST-PC1/PC2 fusion protein. Furthermore, avidin-FITC or avidin-Texas Red, when added alone, did not contribute to background staining.

Results from colabeling studies matched those from single-staining experiments. Examination for coexistence of glucagon with PC2 revealed that both were present within the same subcellular compartment (Fig. 4, C and D, respectively). The glucagon-specific fluorescence was notably more intense than the biotin-avidin-amplified PC2 staining, suggesting that glucagon is much more abundant in granules than PC2. Control incubations containing only the EM7 primary antiserum and both secondary antisera showed no nonspecific interactions of the Texas Red-conjugated secondary antiserum with the PC2 primary antiserum when observed using the rhodamine cube, yet demonstrated punctate PC2-like immunoreactivity when viewed through the FITC cube. Likewise, controls treated with only the glucagon primary antiserum and both secondary antisera demonstrated no nonspecific binding of the PC2 secondary antisera with the glucagon primary antiserum with the FITC cube, whereas glucagon immunoreactivity was maintained when viewed through the rhodamine cube. The narrow band-pass filters for both the rhodamine and FITC cubes used prevented any interfering spectral emission from either FITC or Texas Red.

Peptide Mapping Analysis

Mouse glucagon-related peptides were metabolically labeled with [H]tryptophan and separated by RP-HPLC. The identity of peptides having retention times identical to those of known proglucagon cleavage products was verified by RIA and peptide mapping. TC1-6 cells were pulse-labeled for 90 min, and extracted proteins were separated by RP-HPLC. Fig. 6( upper panel) depicts the elution profile of proglucagon and its cleavage products. All labeled peptides that eluted coincident with synthetic glucagon-related peptides were digested with trypsin and then with CPB. The four peptides that eluted with retention times of 54/55, 56, 58, and 60 min (Fig. 6, upper panel) were also treated. Fig. 2B shows the sequences of the tryptic/CPB fragments from proglucagon that contain tryptophan. The peptide mapping analyses revealed that the predominant radiolabeled peaks coeluted with the tryptophan-containing tryptic fragments of glucagon (Fig. 6 A, peak 1), GLP-I (Fig. 6 B, peak 2), and GLP-II (Fig. 6 C, peak 3). It was also observed that peaks C1, C2, and C3 (Fig. 6, upper panel) each yielded all three tryptophan-containing peptides, suggesting that these extract peptides represent either proglucagon or a modified form of proglucagon. The 60-min peak (Fig. 6 D) yielded only the tryptic fragments of GLP-I and GLP-II and is thus identified as MPGF. Peptide mapping was performed at least three times on each peptide; the chromatograms in Fig. 6depict representative results.


Figure 6: Peptide mapping of mouse proglucagon and proglucagon products. Upper panel, chromatogram of the extract from a 90-min pulse labeling. The arrowheads indicate fractions (±1) taken for trypsin and carboxypeptidase B treatment. The acetonitrile gradient used is signified by the dashed line. A depicts the elution pattern of the trypsin/carboxypeptidase B products of all three extract peaks labeled A in the upper panel and shows the acetonitrile gradient used ( dashed line) for all peptide mapping. B-D show the representative peptide mapping results from extract peaks B-D. In A-D, the numbers 1-3 indicate the elution positions of the tryptophan-containing trypsin/carboxypeptidase B fragments of glucagon, GLP-I, and GLP-II, respectively.



In Vitro Conversion Analyses

To determine a potential role for PC1 and/or PC2 in proglucagon processing, HPLC-purified [H]tryptophan-labeled proglucagon and MPGF were incubated with recombinant PC1 and immunopurified PC2 in vitro, and the cleavage products were separated by HPLC. Products were identified based on their retention times as indicated in Fig. 6 ( upper panel). Under the conditions employed, either PC1 or PC2 alone proteolytically cleaved proglucagon to yield MPGF, glicentin, and oxyntomodulin, while only PC1 was capable of releasing GLP-I from MPGF (Fig. 7). Neither PC1 nor PC2 (nor a combination of the two) processed glucagon from proglucagon in vitro. MPGF was proteolytically cleaved by PC1, but not by PC2, to yield GLP-I (Fig. 8). Mouse GLP-II may also be a product of in vitro processing by PC1 alone or in concert with PC2; however, only small amounts of GLP-II-like peptide products were generated in any of the experiments performed. There was no evidence of nonspecific degradation of MPGF, glicentin, oxyntomodulin, or GLP-I. Therefore, it is improbable that nonspecific degradation contributed to the absence of labeled glucagon in all of the experiments in which the recombinant convertases were incubated with proglucagon.


Figure 7:In vitro cleavage of mouse proglucagon by recombinant PC1 and/or immunopurified PC2. A, proglucagon peak that eluted with a retention time of 58 min ( ProGlgn) incubated with carboxypeptidase B ( CPB) alone (acetonitrile gradient is indicated by the dashed line); B, precursor incubated alone and in the presence of recombinant PC1; C, precursor incubated alone and in the presence of immunopurified PC2; D, precursor incubated alone and in the presence of both PC1 and PC2. The arrowheads correspond to the elution positions of the following: 1, glicentin; 2, oxyntomodulin; 3, glucagon; 4, GLP-I-(1-36)-amide/GLP-I-(7-36)-amide; 5, proglucagon that eluted with a retention time of 54/55 min; 7, proglucagon that eluted with a retention time of 58 min; 8, MPGF. In B-D, the solid line indicates profiles obtained from control incubations (no enzyme), and the dashed line depicts profiles obtained from incubating precursor with enzyme(s). The chromatograms each show representative results from at least three different experiments.




Figure 8:In vitro cleavage of mouse MPGF by recombinant PC1 and/or immunopurified PC2. A, MPGF incubated with carboxypeptidase B ( CPB) alone (acetonitrile gradient is indicated by the dashed line); B, MPGF incubated alone and in the presence of recombinant PC1; C, MPGF incubated alone and in the presence of immunopurified PC2; D, MPGF incubated alone and in the presence of both PC1 and PC2. The arrowheads correspond to the following: 4, GLP-I-(1-37)-amide; 8, MPGF. The solid line indicates profiles obtained from control incubations (no enzyme), and the dashed line depicts profiles obtained from incubating precursor with enzyme(s). The chromatograms each show representative results from at least three different experiments.




DISCUSSION

In this study, we have continued the characterization of the TC1-6 cell line by amplifying and sequencing the mouse preproglucagon cDNA, previously unknown, and by developing an HPLC-based in vitro conversion assay for PCs using radiolabeled proglucagon. The mouse preproglucagon sequence provides an excellent model to use in studying PC specificity because of its high conservation and the observation that it is differentially processed in various tissues. The amino acid sequences of mouse glucagon and GLP-I are 100% homologous when compared with the published sequences of rat and human preproglucagons. This is not surprising in view of the well known roles of glucagon and GLP-I as physiological mediators.

Using primers directed toward nonhomologous regions of mouse PC1 and PC2, we amplified PCR products from TC1-6 mRNA. The PC2 product was present in relatively high abundance, while the PC1 product was barely detectable. After subcloning and sequence confirmation, the PCR products were used as probes for Northern blot analyses. Our results confirm the findings of Neerman-Arbez et al. (41) that both PC1 and PC2 are present within these cells. The presence of PC1 mRNA was only detected after prolonged exposure, which could explain why Rouillé et al. (42) did not detect any PC1 mRNA in their studies of TC1-6 cells. We also found that PC1 is expressed in TC1-6 cells by Western (data not shown) and immunocytochemical (Fig. 5) analyses. The relative abundance and size of both PC1 and PC2 mRNAs found in the TC1-6 tumor cell line closely resemble the sizes of PC1 and PC2 mRNAs extracted from normal intact islets (19, 41) .


Figure 5: Immunocytochemical detection of PC1 in TC1-6 cells in a single-labeling experiment. The arrows indicate areas of punctate immunostaining. Bar, 10 µm.



The presence of both PC1 and PC2 in TC1-6 cells was verified immunocytochemically. PC2 and glucagon were shown to coexist in punctate structures assumed to be secretory granules. Positive control experiments showed both PC1 and PC2 to be present within the rat pituitary gland (data not shown), which supports in situ hybridization data obtained from rat (26) and mouse (51) . In TC1-6 cells, PC1 immunostaining appeared in a fine punctate pattern with a significantly lower staining intensity compared with PC2 or glucagon. These results are consistent with previously reported Northern and Western blot analyses (40, 41, 42) . The fine granular appearance of the PC2-like staining compared with the intense coarse staining of glucagon in TC1-6 cells may reflect the proportion of enzyme relative to substrate present within these cells. These results corroborate the findings of Rouillé et al. (42) , Marcinkiewicz et al. (52) , and Nagamune et al. (53) , who used intact mouse and rat islet tissue to demonstrate immunological colocalization of glucagon and PC2 in pancreatic -cells. Our results extend their findings by clearly demonstrating glucagon and PC2 colocalization in TC1-6 secretory granules.

HPLC and peptide mapping studies performed on metabolically labeled TC1-6 peptides verified the observations of other investigators that TC1-6 cells process proglucagon in much the same manner as pancreatic -cells. Pulse-chase analyses in our laboratory confirmed the results of Rouillé et al. (42) . MPGF, one of the predominant products found in pancreatic -cells, appears to be the first cleavage product, followed by glicentin and oxyntomodulin, glucagon, and finally GLP-I in the TC1-6 cell line (data not shown). Holst et al. (54) , using HPLC, RIA, and mass spectrometry analyses of human and porcine pancreatic -cells, revealed that MPGF is found in amounts nearly equimolar to glucagon. In TC1-6 cells, MPGF is the predominant product after 90 min of chase, which is similar to the findings of Holst et al. (54) . Our HPLC elution protocol detected three proglucagon peaks eluting with retention times of 54/55, 56, and 58 min, respectively. All three peaks were confirmed as mouse proglucagon by peptide mapping studies. It is possible that the peaks at 54/55 and 56 min are differentially sulfoxidized forms of the peak at 58 min. Studies to determine whether this is an accurate supposition are currently in progress.

To determine if PC1, PC2, or both are potential physiological mediators of proglucagon processing, we performed in vitro assays in which radiolabeled proglucagon and the enzymes were incubated together. Results from these assays indicate that both PC1 and PC2 proteolytically cleaved proglucagon to yield MPGF, glicentin, and oxyntomodulin, while PC1 alone was capable of releasing GLP-I from MPGF. Neither PC1 nor PC2 processed glucagon from proglucagon in vitro. There are several possible explanations for the observation that radiolabeled glucagon was not detected as a cleavage product in our in vitro assays. First, it is possible that glucagon may indeed be released, but in very small quantities, resulting in the release of amounts of labeled peptide that do not exceed background levels. Another explanation may be that optimal conditions for cleaving at the Lys-Arg site at the C terminus of glucagon may not have been achieved under the assay conditions employed. However, neither of these suggestions seems reasonable because other readily identifiable cleavage products were detected in all experiments performed. It is also possible that glucagon itself may be nonspecifically cleaved. This seems highly unlikely due to the consistent generation of other proglucagon products that did not exhibit random degradation. It is improbable that sulfoxidation of methionine residues in proglucagon could have completely prevented cleavage at the C terminus of glucagon because the precursor that is most hydrophobic (the peak eluting with a retention time of 58-59 min in Figs. 6 and 7), and therefore the least likely to contain any methionine sulfoxide, yielded no glucagon after incubations with PC1, PC2, or both (three separate experiments for each combination). It is emphasized that all of the in vitro incubations were replicated with two independently prepared lots of recombinant PC1 and PC2 and that, prior to use, each batch of PC1 or PC2 was tested for activity with a fluorogenic substrate. As a result of all these considerations, we conclude that, in vitro under the conditions employed, neither PC1 nor PC2 cleaves proglucagon at the C-terminal Lys-Arg site required for the release of glucagon.

We are particularly intrigued by the inability of PC2 to mediate cleavage of glucagon from proglucagon. The Lys-Arg site, located between the C-terminal peptide and the A-chain of proinsulin, is thought to be cleaved by PC2 (55, 56, 57, 58) . Moreover, PC2 is the predominant convertase present in the TC1-6 cell line (Refs. 40-42 and this study) and in intact islet -cells (42, 52) . In the mouse proglucagon sequence, there are three Lys-Arg sites: at the N terminus of glucagon/oxyntomodulin, at the C terminus of glucagon, and at the N terminus of MPGF. Rouillé et al. (42) , using transient transfection of mammalian expression vectors encoding antisense mRNA directed toward PC2, concluded that a reduction in PC2 expression resulted in a decrease in the processing of glucagon from proglucagon in TC1-6 cells. Our in vitro results do not corroborate their findings. While it is important to note that the conditions of our in vitro assay do not completely mimic the environment within secretory granules in the intact cell, the fact remains that two of the three available Lys-Arg cleavage sites in proglucagon were routinely processed in our experiments. An additional consideration is that there may be components within the secretory granule that either augment or inhibit the processing of precursors in living cells. A known modulator of PC2 activity is 7B2, which is found in relatively high concentrations in the TC1-6 cell line.() Considering the fact that 7B2 is an endogenous PC2 inhibitor (59) , the potential role of PC2 in processing proglucagon may be modulated by interaction with 7B2. In the context of our studies, it is important to note that PC2 did not cleave glucagon from proglucagon even in the absence of 7B2. There is also at least one other PC (PC6A) that is expressed in the TC1-6 cell line (40) that may play a role in processing proglucagon in these cells. Further investigation will be necessary to determine the roles of 7B2 and PC6A, if any, in modulating or mediating the processing of the mouse glucagon precursor. It will also be necessary to obtain a more complete definition of the secretory granule milieu to fully understand the mechanisms regulating PC activity in the intact cell.

Fig. 9 is a model of mouse proglucagon processing that was developed based on the data obtained from our in vitro studies. Either PC1 or PC2 can mediate the first cleavage that occurs at the Lys-Arg site that links glicentin and MPGF. Both enzymes can also cleave at the Lys-Arg site N-terminal to glucagon, yielding oxyntomodulin. Under the conditions employed, neither PC1 nor PC2 can process at the Lys-Arg site at the C terminus of glucagon that would result in the formation of glucagon from oxyntomodulin. Only PC1 can cleave at the Arg-Arg site that results in the release of GLP-I from MPGF. The determination of which of the PCs is responsible for mediating cleavage of glucagon from proglucagon will require further investigation.


Figure 9: Model of mouse proglucagon processing by recombinant PC1 and immunopurified PC2. Potential sites of processing not observed to be utilized in the in vitro assays are indicated with question marks. See the legend to Fig. 1 for other details.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 44986 (to B. D. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Emory University, 1648 Pierce Dr., Atlanta, GA 30322. Tel.: 404-727-6251; Fax: 404-727-6256.

The abbreviations used are: PCs, prohormone convertases (the prefix ``h'' stands for human); GLP-I and -II, glucagon-like peptide-I and -II (the prefix ``h'' stands for human); MPGF, major proglucagon fragment; RIA, radioimmunoassay; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s); FITC, fluorescein isothiocyanate; RP-HPLC, reverse-phase high pressure liquid chromatography; GST, glutathione S-transferase; CPB, carboxypeptidase B.

I. Lindberg, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Nabil Seidah for the generous gift of the mouse PC1 and PC2 full-length cDNA plasmids and Dr. Douglas Hanahan for the TC1-6 cells.


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