From the
The mouse tumor cell line
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)
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
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.
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.
In this study, we have continued the characterization of the
Using
primers directed toward nonhomologous regions of mouse PC1 and PC2, we
amplified PCR products from
HPLC and peptide mapping studies performed on
metabolically labeled
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
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.
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)(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) .
-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 GenBank
Data 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.
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 INV
F` 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
10
cells) 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
10
cpm/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.
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-cm
flasks (Falcon) until reaching 80% confluency at
2
10
cells. 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 C
cartridge (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 C
column (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 CaCl
and 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.
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.
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.
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.
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.
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.
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.
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.
TC1-6 cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.