(Received for publication, July 13, 1995; and in revised form, August 17, 1995)
From the
Proglucagon is processed differently in the islet cells
and the intestinal endocrine L cells to release either glucagon or
glucagon-like peptide 1-(7-37) (GLP1-(7-37)), peptide
hormones with opposing actions in vivo. In previous studies
with a transformed
cell line (
TC1-6) we demonstrated that
the kexin/subtilisin-like prohormone convertase, PC2 (SPC2), is
responsible for generating the typical
cell pattern of
proglucagon processing, giving rise to glucagon and leaving unprocessed
the entire C-terminal half-molecule known as major proglucagon fragment
or MPGF (Rouillé, Y., Westermark, G., Martin, S.
K., Steiner. D. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,
3242-3246). Here we present evidence, using mouse pituitary
AtT-20 cells infected with a vaccinia viral vector encoding
proglucagon, that PC3 (SPC3), the major neuroendocrine prohormone
convertase in these cells, reproduces the intestinal L cell processing
phenotype, in which MPGF is processed to release two glucagon-related
peptides, GLP1 and GLP2, while the glucagon-containing N-terminal
half-molecule (glicentin) is only partially processed to oxyntomodulin
and small amounts of glucagon. Moreover, in AtT-20 cells stably
transfected with PC2 (AtT-20/PC2 cells), glicentin was efficiently
processed to glucagon, providing further support for the conclusion
that PC2 is the enzyme responsible for the
cell processing
phenotype. In other cell lines expressing both PC2 and PC3 (STC-1 and
TC-3), proglucagon was also processed extensively to both glucagon
and GLP1-(7-37), although STC-1 cells express lower levels of PC2
and processed the N-terminal domain to glucagon less efficiently. In
contrast, GH
C
and COS 7 cells, which express
very little or no PC2 or PC3, failed to process proglucagon, aside from
a low level of interdomain cleavage which occurred only in the
GH
C
cells. In vitro PC3 did not cleave
at the single Arg residue in GLP1 to generate GLP1-(7-37), its
truncated biologically active form, indicating the likelihood that
another convertase is required for this cleavage.
The discovery of a novel family of mammalian proteases related
to the yeast processing enzyme kexin and the bacterial serine protease
subtilisin has provided new insights into the cellular mechanisms of
precursor processing in the secretory
pathway(1, 2, 3) . Recent studies have
suggested that two members of this six-member family, PC2 (SPC2) and
PC3 (SPC3; also called PC1), are the key enzymes involved in the
proteolytic processing of a large variety of neuroendocrine precursors
in the brain and many endocrine tissues throughout the body in which
these enzymes are predominantly localized(1, 3) . PC2
and PC3 both participate in the processing of proinsulin to insulin in
the pancreatic cell (4) and of POMC (
)to ACTH
and/or other products in the pituitary(5, 6) .
Moreover, it has been demonstrated that the known differential
processing of POMC to different products in the anterior versus intermediate lobes of the pituitary (7) is due to the
differential expression of PC2 and PC3 in these lobes (8) .
Attention is thus focused on defining the potential role of these
and/or other cellular proteases in the differential processing of other
important neuroendocrine precursors such as proglucagon.
The 18-kDa
mammalian proglucagon protein contains three homologous hormonal
sequences, glucagon, glucagon-like peptide 1 (GLP1), and glucagon-like
peptide 2 (GLP2), separated by two intervening peptides, IP-1 and IP-2,
and preceded by an N-terminal extension called glicentin-related
polypeptide (GRPP) (Fig. 1). These peptides are all linked by
pairs of basic amino acids (Lys-Arg or Arg-Arg), that are used as
cleavage sites during the processing. In mammals, the same precursor is
initially synthesized in the cells of the islets of Langerhans in
the pancreas and in the endocrine L cells of the intestinal
mucosa(9, 10) ; however, differential processing
results in the formation of different sets of peptides with opposing
biological activities. The pancreatic
cell secretes glucagon,
which stimulates glycogenolysis and gluconeogenesis in the liver and
counterbalances the hypoglycemic action of insulin, whereas the
intestinal L cell secretes a very potent insulinotropic hormone,
recently identified as GLP1-(7-37), a truncated form of
GLP-1(11, 12) .
Figure 1:
Proglucagon
differential processing in the pancreas and the intestine. The primary
structure of proglucagon is represented with glucagon, glucagon-like
peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2) sequences in hatched boxes. The upper
part shows the peptides resulting from proglucagon processing in
the pancreatic cell. These peptides are the glicentin-related
polypeptide (GRPP, proglucagon-(1-30)), glucagon
(proglucagon-(33-61)), the intervening peptide 1 (IP-1,
proglucagon-(64-69)), and the major proglucagon fragment (MPGF, proglucagon-(72-158)). MPGF is partially
processed to GLP-1 (proglucagon-(72-107)). The lower part shows the peptides resulting from proglucagon processing in the
intestinal L cell. These peptides are glicentin
(proglucagon-(1-69)), truncated GLP-1 (tGLP-1,
proglucagon-(78-107)), intervening peptide 2 (IP-2,
proglucagon-(111-122)), and GLP-2 (proglucagon-(126-158)). Glicentin is partially processed to
GRPP and oxyntomodulin (PG 33-69). The cleaved sites are
indicated by arrows, with the sequence surrounding the site shown above
or below the arrow. Partially processed sites are indicated by dashed arrows. Note that the monobasic cleavage at Arg
occurs only in the intestinal L cell.
The pancreatic cell processes
proglucagon mainly to glucagon, GRPP, IP-1, and MPGF
(proglucagon-(72-158)), a 10-kDa peptide encompassing the GLP-1,
IP-2, and GLP-2 sequences(13, 14) . A small fraction
(10 to 20%) of the MPGF is further processed to GLP-1(15) . Low
amounts of glicentin (proglucagon-(1-69)) have also been detected
in the pancreas (16) , suggesting that it is an intermediate in
the formation of glucagon. This processing pathway has been
demonstrated recently through pulse-chase experiments in
TC1-6
cells, an islet-derived cell line transformed with SV40 large T
antigen(17) . Proglucagon is initially cleaved at the
Lys
-Arg
site to produce glicentin and MPGF.
Glicentin is later processed at the Lys
-Arg
and Lys
-Arg
sites to yield GRPP,
glucagon, and IP-1, while MPGF accumulates and is only slowly and
partially processed to GLP-1-(1-36)-amide
(proglucagon-(72-107)), after cleavage at the
Arg
-Arg
site. Thus, the pancreatic
processing of proglucagon occurs with an initial interdomain cleavage,
followed by efficient processing of the N-terminal domain (glicentin)
and very little processing of the C-terminal domain (MPGF).
In
contrast, in the intestinal L cells, proglucagon processing results in
the efficient formation of GLP-1, IP-2, and GLP-2(18) .
Glucagon levels are very low in the intestine(19) . Rather, the
N-terminal domain remains incompletely processed in the form of
glicentin (20) and is only partially cleaved into GRPP and
oxyntomodulin (proglucagon-(33-69))(21) , a form of
glucagon C-terminally extended with IP-1(22) . An additional
cleavage occurs at a single arginine residue within the GLP-1 sequence
(Arg), yielding shortened active forms of GLP-1,
GLP-1-(7-37) (proglucagon-(78-108)) and its desglycyl,
C-terminally amidated counterpart, GLP-1-(7-36)-amide
(proglucagon-(78-107)), collectively known as truncated GLP-1, or
tGLP-1(12) . The processing of proglucagon in intestinal L
cells thus may involve an initial interdomain cleavage, probably at the
same site as observed in the pancreas, and is followed by extensive
processing of only the C-terminal domain.
The simplest explanation
for the alternative processing of the N- or C-terminal domains of
glucagon, after their cleavage at the interdomain processing site,
would be that it is due to the expression of different convertases in
the and L cells. This view is supported by recent findings that
pancreatic
cells express high levels of PC2 and low levels, if
any, of PC3(17, 23, 24) , whereas intestinal
L cells contain immunoreactive PC3 but not PC2(25) . This
suggests that PC2 generates the pancreatic phenotype while PC3 may be
largely responsible for the intestinal phenotype. This hypothesis is
also supported by the results of the present study correlating
proglucagon processing in cell lines with differing levels of
endogenous PC2 and PC3 convertases. We have examined the processing of
proglucagon in a number of endocrine and non-endocrine-derived cell
lines and have correlated the observed processing patterns with the
levels of expression of the prohormone convertases PC2 and PC3. The
results support the conclusions that PC3 is responsible for the
processing of the C-terminal domain of proglucagon to release GLP1, and
they indicate that another, as yet unidentified protease may be
required for the conversion of GLP1 to tGLP1.
For some experiments with
TC3 and COS 7 cells, the cell pellet was resuspended in 1 M acetic acid and extracted for 10 min at 95 °C. The extract was
concentrated in a SepPak C18 cartridge (Millipore), and the peptides
were eluted with 60% acetonitrile, 0.1% trifluoroacetic acid. The
peptide extract was freeze-dried, redissolved in 20 µl of water,
adjusted to 200 µl with immunoprecipitation buffer, incubated for
30 min on ice, and centrifuged for 15 min at 15,000
g to remove insoluble materials. The whole supernatant was used for
immunoprecipitation. Control experiments with
TC1-6 cells showed a
similar recovery of the glucagon-containing peptides with both
extraction methods (not shown). Recovery of
I-labeled
glucagon by these methods was greater than 80%.
Figure 2:
Identification of the
proglucagon-derived peptides produced in AtT-20 cells. AtT-20 cells
were infected with VV:GLU (lanes 1 and 4) or the
control VV:WT (lanes 2 and 3) virus, labeled with
[H]Phe (lanes 1 and 2) or
[
S]Met (lanes 3 and 4) for 12
h. Immunoprecipitations were performed on cell lysates with antibodies
directed against glucagon (lanes 3 and 4) or against
GLP-1 (lanes 1 and 2). Immunoprecipitates were
analyzed by SDS-PAGE. The migration of the molecular mass markers as
well as proglucagon-derived peptides is indicated. PRO,
proglucagon; GLI, glicentin (proglucagon-(1-69)); OXT, oxyntomodulin (proglucagon 33-69); GLU,
glucagon (proglucagon 33-61); MPGF, major proglucagon
fragment (proglucagon-(72-158/160)); GLP-1, N-terminally
extended glucagon-like peptide 1 (proglucagon-(72-108) and/or
proglucagon-(72-107)-amide); tGLP-1, truncated
glucagon-like peptide 1 (proglucagon-(78-108) and/or
proglucagon(78-107)-amide).
The identification of MPGF as a processing
intermediate was also supported by its detection in the conditioned
medium of infected cells. In the absence of added secretagogue,
infected cells secreted large amounts of proglucagon, glicentin, and
MPGF (Fig. 3). Shorter peptides, that could be
immunoprecipitated from the cell lysates, were not detected in the
medium. It is a well established observation that AtT-20 cells and
other transformed endocrine cells in culture constitutively secrete
large amounts of unprocessed precursors and intermediates resulting
from the earliest cleavage step(26) . Thus, the finding of
secreted material cleaved at the interdomain
Lys-Arg
site suggests that this is the first
cleavage during proglucagon processing in AtT-20 cells, as has also
been demonstrated in
TC1-6 cells(17) . Following this
cleavage, the N-terminal domain (glicentin) is only partially converted
to oxyntomodulin, whereas the C-terminal domain (MPGF) is extensively
processed to the 3.4-kDa GLP-1, probably via 4-kDa GLP-1 as an
intermediate.
Figure 3: Identification of the proglucagon-derived peptides secreted by AtT-20 cells under unstimulated conditions. AtT-20 cells were infected and labeled as in Fig. 2. Immunoprecipitations were carried out on the media, with antibodies directed against glucagon (lanes 3 and 4) or against GLP-1 (lanes 1 and 2). Immunoprecipitates were analyzed by SDS-PAGE. The migration of the molecular mass markers as well as proglucagon-derived peptides is indicated. Abbreviations are as in Fig. 2.
The putative 3.4-kDa GLP-1 was further characterized
by radiosequencing of the [H]Phe-labeled peptide,
purified by immunoprecipitation with the carboxyamidation-specific
antiserum 89-390, followed by HPLC. Two peaks of radioactivity
indicated the presence of Phe residues in positions 6 and 22 of the
peptide (Fig. 4). This result demonstrates that the 3.4-kDa
GLP-1 produced in AtT-20 cells is indeed GLP-1-(7-36)-amide or
tGLP-1. This result also implies that a cleavage at the monobasic
Arg
site had occurred during the processing of proglucagon
in AtT-20 cells, in addition to the other observed cleavages, that all
occurred at dibasic sites.
Figure 4:
Identification by radiosequencing of the
tGLP-1 produced in AtT-20 cells. GLP-1 was immunoprecipitated with
carboxyamidation specific antiserum 89-390 from a lysate of
AtT-20 cells infected with VV:GLU and radiolabeled with
[H]Phe. The immunoprecipitated material was
further purified by reverse-phase HPLC and submitted to 30 cycles of
Edman degradation. Anilinothiazolinone derivatives were collected at
each cycle and assayed for radioactivity in a liquid scintillation
counter. The sequence of proglucagon-(78-107) is shown under
the graph.
Figure 5:
Identification of the glucagon-containing
peptides produced in AtT-20/PC2 cells. AtT-20/PC2 (lanes 1 and 2) or wild type AtT-20 cells (lanes 3 and 4)
were infected with VV:GLU (lanes 2 and 4) or the
control VV:WT (lanes 1 and 3) virus and labeled with
[S]Met for 12 h. Glucagon-containing peptides
were immunoprecipitated from the cell lysates and analyzed by SDS-PAGE. Abbreviations are as in Fig. 2.
Figure 6:
Immunoblot analysis of the expression of
PC2 and PC3 in four endocrine cell lines. 25 µg of proteins of a
crude granular fraction of TC1-6 (lanes 1 and 5),
TC3 (lanes 2 and 6), AtT-20 (lanes 3 and 7), and STC1 cells (lanes 5 and 8) were resolved in SDS-PAGE and electrophoretically
transferred onto an Immobilon P membrane. The blot was developed using
the PC3 antiserum 2B6 (lanes 1-4) or PC2 antiserum
PC2pep4 (lanes 5-8).
The processing of the
N-terminal domain of the precursor in STC-1 cells was analyzed by
immunoprecipitation with the glucagon antibody. Three major
glucagon-containing peptides of 9, 7.5, and 3.4 kDa were found (Fig. 7). Based on their apparent molecular masses, and on their
strong immunoreactivity toward the C-terminal specific glucagon
antiserum P7 (not shown), the 3.4-kDa and 7.5-kDa peptides were
identified as glucagon and proglucagon-(1-61) (GRPP linked to
glucagon, see Fig. 1), respectively. The 9-kDa peptide was
identified as the glicentin. A minor component of 4.5-kDa was also
detected (Fig. 7) that was not immunoprecipitated with the P7
antiserum (not shown). This peptide was therefore identified as
oxyntomodulin. These four glucagon-containing peptides have been
identified previously in TC1-6 cells(17) .
Figure 7:
Proglucagon processing in STC1 cells. STC1
cells were labeled with [H]Phe for 12 h.
Immunoprecipitations were performed on cell lysates with antibodies
directed against glucagon (lanes 2) or against the C-terminal
sequence of amidated GLP-1 (lane 1). Immunoprecipitates were
analyzed by SDS-PAGE. The migration of the molecular mass markers as
well as proglucagon-derived peptides is
indicated.
The processing of the C-terminal domain was analyzed by immunoprecipitation with GLP-1 antisera. The carboxyamidation-specific antiserum 89-390 immunoprecipitated a 4-kDa and a 3.4-kDa peptide, the latter being much more abundant than the former (Fig. 7). Similar experiments with the GLP-1 antiserum 2135 also immunoprecipitated these two peptides altogether with small amounts of the 19-kDa proglucagon and of the 8-kDa MPGF (not shown). On the other hand, antiserum 165-3, which is specific for the N-terminal extension of GLP-1-(1-37), immunoprecipitated the 19-, 8-, and 4-kDa GLP-1-containing peptides, but failed to react with the 3.4-kDa peptide (not shown). Taken together, these results suggest that the C-terminal domain of proglucagon is efficiently processed to tGLP-1, whereas the processing of the N-terminal domain to glucagon is incomplete in STC-1 cells, as previously shown by Blache et al.(33) .
The analysis of the processing of proglucagon
expressed in TC-3 cells after infection with VV:GLU was
complicated by two problems. First, infection of this cell line with
the recombinant viral vector was found to result in a much lower
expression of proglucagon than in the other cell lines used in this
study. Second, very similar results were obtained after infection with
VV:GLU and with the control wild type virus, at low multiplicity of
infection (not shown). We reasoned from these observations that
TC-3 cells are probably poorly infected by vaccinia virus and that
this cell line is also endogenously expressing proglucagon at a low
level, thus resulting in similar patterns of immunoprecipitated bands
after infection with the recombinant VV:GLU or the control virus.
Endogenous proglucagon expression in
TC-3 cells was confirmed by
RT-PCR (data not shown). Then, using a higher multiplicity of infection
and a longer time of contact with the virus, a clear overexpression of
proglucagon was observed in the VV:GLU infected cells compared to the
control cells infected with the wild type virus, as shown in Fig. 8. Glucagon-containing peptides were immunoprecipitated
from media and cell extracts after concentration on SepPak cartridges.
The 19-kDa precursor and the 3.4-kDa glucagon were detected in the
cells infected with VV:GLU, whereas only glucagon was
immunoprecipitated from the cells infected with the control VV:WT. The
higher amounts of glucagon detected after infection with the
recombinant virus indicated that both the endogenously expressed
proglucagon and the precursor produced by the viral vector are
processed to glucagon. The accumulation of unprocessed precursor after
recombinant vaccinia virus infection has often been noted by
others(5, 34) . On the other hand, the apparent
absence of intracellular accumulation of processing intermediates
suggests that proglucagon is efficiently processed to glucagon in
TC-3 cells. This apparent absence of intermediates could also
reflect a pathway of processing different from the one observed in
TC1-6 cells, but this alternative is less likely, since a 9-kDa
glicentin was detected in the medium (Fig. 8), suggesting that
it is an intermediate in the processing of proglucagon to glucagon in
TC-3 cells, as it is in
TC1-6 cells(17) . Similarly,
only proglucagon and tGLP-1 sized peptides were detected after
immunoprecipitation with GLP-1 antiserum (not shown). All these results
indicate that
TC-3 cells endogenously express proglucagon and
process it both to glucagon and to tGLP-1. Thus, in contrast to
TC1-6 cells and AtT-20 cells, which express either PC2 or PC3 and
process proglucagon either to glucagon or to tGLP-1, respectively,
STC-1 and
TC-3 cells, which express both PC2 and PC3, are able to
process proglucagon to both glucagon and tGLP-1.
Figure 8:
Proglucagon processing in TC3 cells.
TC3 cells were infected with VV:GLU (lanes 2 and 4) or the control VV:WT (lanes 1 and 3)
virus, labeled with [
S]Met for 12 h.
Immunoprecipitations were performed on cell lysates (lanes 3 and 4) and on medium (lanes 1 and 2)
with antibody directed against glucagon. Immunoprecipitates were
analyzed by SDS-PAGE. The migration of the molecular mass markers as
well as proglucagon-derived peptides is indicated. Abbreviations are as in Fig. 2.
Infection of
GHC
cells with VV:GLU resulted in the
production of mainly unprocessed precursor (Fig. 9). The
glucagon antibody immunoprecipitated large amounts of 19-kDa
proglucagon and small amounts of 9-kDa glicentin, and the GLP-1
antiserum immunoprecipitated the precursor and small amounts of 8-kDa
MPGF. Immunoprecipitations from the medium resulted in a very similar
pattern (not shown). These results suggest that GH
C
cells, although able to store a fraction of the expressed
proglucagon in an intracellular compartment, are unable to efficiently
process it, as previously reported by Drucker et
al.(36) . The only partial cleavage observed occurred at
the interdomain Lys
-Arg
site and may have
been due to the presence of low levels of PC2, PC3, or other proteases.
Figure 9:
Proglucagon processing in
GHC
cells. GH
C
cells
were infected with VV:GLU and labeled for 12 h. Immunoprecipitations
were performed on cell lysates with antibodies directed against
glucagon (lane 2) or against GLP-1 (lane 1).
Immunoprecipitates were analyzed by SDS-PAGE. The migration of the
molecular mass markers as well as proglucagon-derived peptides is
indicated.
The lack of processing was even more obvious with COS 7 cells. After infection with VV:GLU and radiolabeling, we were unable to immunoprecipitate any proglucagon or proglucagon-derived peptides from cell extracts, even after concentration on SepPak cartridge (not shown). However, immunoprecipitation from the medium revealed the presence of large amounts of proglucagon (Fig. 10). No smaller forms were detected even after prolonged exposure. These results indicate that COS 7 cells rapidly secrete proglucagon, without being able to process it at any of its potential cleavage sites, despite the presence of furin in these cells(37) .
Figure 10:
Proglucagon processing in COS 7 cells.
COS 7 cells were infected with the wild type virus (lane 1),
or the VV:GLU, and labeled with [S]Met for 12 h.
Media were concentrated and immunoprecipitated with the glucagon (lanes 1 and 2) or GLP-1 (lane 3)
antibody.
The results of the
various expression experiments in the cell lines are summarized in Table 1. A good correlation between PC2 expression and ability to
process proglucagon to glucagon (efficient cleavage at both
Lys-Arg
and Lys
-Arg
sites) can be observed. Similarly, expression of PC3 correlates
with processing to tGLP-1 (efficient cleavage at both Arg
and Arg
-Arg
sites).
Figure 11:
In vitro activity of recombinant PC3 on
GLP-1 and proinsulin. I-labeled proinsulin (A)
and GLP-1 (B) were used as substrates for in vitro conversion studies performed with recombinant PC3, obtained from
concentrated medium of VV:PC3-infected COS 7 cells (lanes 3 and 6). Medium of VV:WT-infected COS 7 cells was used as
a negative control (lanes 2 and 5). Incubations
without enzyme are shown in lanes 1 and 4. Reactions
were incubated for 16 h at 32 °C, stopped by boiling, concentrated,
and analyzed by SDS-PAGE under reducing conditions and autoradiography.
Similarly, digestion of
I-labeled GLP-1 by endoproteinase
Arg-C yield a 3.4-kDa peptide, having the same migration as
GLP-1-(7-36)-amide (lane 7). Designations are: Pro, intact proinsulin; C-A, C-peptide-A chain
fragment; B chain, B chain Arg-Arg; 1-36,
GLP1-(1-36)-amide; 7-36,
GLP1-(7-36)-amide.
We have examined the processing of proglucagon in AtT-20 cells after expression with a recombinant vaccinia virus vector. We have shown that this processing is closely similar to that observed in intestinal L cells in that the C-terminal domain is efficiently converted to tGLP-1 (12) while the N-terminal domain is only partially cleaved to oxyntomodulin and very low amounts of glucagon(19) . These results are in good agreement with the recent report by Mineo et al.(38) , who also found that transfected AtT-20 cells process proglucagon to oxyntomodulin to a much greater extent than to glucagon. This resemblance in processing patterns makes AtT-20 cells a good model for studying proglucagon processing in cells with the L cell phenotype and suggests that AtT-20 cells and intestinal L cells are endowed with similar endoproteolytic processing activities.
Of the six SPCs identified thus far, only PC2
and PC3 have been shown to be active in the regulated secretory pathway
of neuroendocrine cells. In AtT-20 cells, PC3 is expressed at a much
higher level than PC2(4) , whereas in TC1-6 cells and
pancreatic
cells, PC2 is present in large excess over
PC3(17, 23, 24) . We have previously shown
that PC2 is the key endoprotease responsible for proglucagon processing
in cells with the
cell phenotype(17) . The results
presented here further support this conclusion and make PC3 a good
candidate for an endopeptidase involved in the processing of
proglucagon in L cells. Indeed, we have shown that only PC3-expressing
cell lines are able to efficiently process the C-terminal domain of the
precursor to tGLP-1. This is further supported by the recent report by
Rothenberg et al.(31) that PC3 is able to cleave in vitro the Lys
-Arg
,
Lys
-Arg
, and Arg
-Arg
processing sites of proglucagon, cleavages that also occur in
AtT-20 cells. Furthermore, Scopsi et al.(25) have
detected immunoreactive PC3, but not PC2 in intestinal L cells. All
these data are compatible with the hypothesis that PC3 is involved in
the processing of proglucagon in L cells.
Just as PC3 expression is
correlated with the ability of a cell line to process the C-terminal
domain to tGLP-1, PC2 appears required for a cell line to be able to
process the N-terminal domain to glucagon. Mineo et al.(38) also have shown that GH and InR1-G9
cells, both expressing PC2, can convert proglucagon to glucagon. This
confirms and extends our previous results with the
TC1-6 cell
line(17) , suggesting that PC2 is the only SPC able to convert
proglucagon to glucagon. However, Rothenberg et al.(31) have recently suggested, on the basis of the result
of an in vitro conversion experiment, that PC2 by itself could
not cleave the Lys
-Arg
processing site at the
C terminus of the glucagon moiety. Accordingly, they concluded that PC2
would only be able to produce oxyntomodulin, and that a second
endopeptidase, possibly PC6, would cleave at the C terminus of glucagon
in
cells. Although this hypothesis can account for the observed
lack of cleavage by PC2 in vitro, it is not compatible with
results from in vivo processing studies. First, the
involvement of PC6 can be ruled out, since
TC3 cells which do not
express detectable levels of PC6 (35) efficiently process
proglucagon to glucagon. Likewise, the glucagon-producing
TC1-6
cells do not express detectable levels of PC6, as assessed by RT-PCR. (
)Similar conclusions can be drawn for all the SPC presently
known, except for PC2, which is the only convertase expressed in all
the cell lines that are able to produce glucagon and only in those cell
lines. Moreover, our results from expression of proglucagon in
AtT-20/PC2 cells leave no doubt concerning the ability of PC2 to cleave
the Lys
-Arg
processing site at the C terminus
of the glucagon moiety in vivo. Perhaps the low activity of
the PC2 preparation used by Rothenberg et al.(31) ,
which only partially cleaved the Lys
-Arg
and
Lys
-Arg
sites, accounts for its failure to
cleave this site in vitro. It is also possible that a factor
present in vivo is missing in the in vitro experiment. On the other hand, some sites appear to be more
``resistant'' to cleavage, possibly due to a less favorable
conformation, and, consequently, the level of expression of the
convertase also has to be taken into consideration. For example, STC-1
cells that express lower levels of PC2 incompletely convert the
N-terminal domain of proglucagon to glucagon. Similarly, limiting
amounts of PC3 only partially convert proinsulin to
des-31,32-proinsulin in vitro(39) , although higher
amounts of PC3 are effective in converting this intermediate to insulin
(see Fig. 11).
The differential processing of proglucagon
underscores the very significant differences in site selectivity
exhibited by PC2 and PC3, as already documented in studies with other
precursors such as POMC (5, 6) and
proinsulin(4) . Although it is now clear that both of these
endoproteases most often cleave prohormones at dibasic sites, the
precise structural basis for their site preferences on their
physiological substrates is still unclear. Thus, in studies on
proglucagon and proinsulin processing PC2 appears to prefer Lys-Arg
sites while PC3 prefers Arg-Arg sites, but this is not the case with
POMC processing, where various Lys-Arg sites are preferentially cleaved
by either PC2 or PC3(5, 6) . Other sequence and/or
conformational elements are thus likely to be crucial determinants of
PC2 or PC3 cleavage motifs. A comparison of the amino acid sequences
surrounding the sites preferred by PC2 or PC3 in proinsulin,
proglucagon, and POMC does not reveal any distinguishing features in
this small sample. Also, very little is known about the conformation of
the polypeptide chain at cleavage sites. It seems possible that
cleavage at one site in a precursor may generate conformational changes
at other sites that in turn would then become better substrates for one
or the other convertase, as suggested in the case of
proinsulin(40) . This idea is further supported by our
observations that in proglucagon cleavages do not occur randomly, but
are temporally ordered. In TC1-6 cells, PC2 cleaves first at
Lys
-Arg
and then at both
Lys
-Arg
and Lys
-Arg
sites in the glicentin produced by the first cleavage, but does
not cleave these sites in intact proglucagon(17) . Similarly in
AtT20 cells, PC3 cleaves first at the Lys
-Arg
site and then is able to further process the C-terminal domain.
Production of bioactive tGLP-1 involves the cleavage of the
proglucagon at Arg, a monobasic processing site.
Interestingly, PC3 has been reported recently to cleave prodynorphin at
a monobasic site(34) . It has been suggested that the lack of a
P2 basic residue is compensated by the presence of a P4
arginine(41) . However, the monobasic cleavage site in
proglucagon has a glutamic residue at this position, a characteristic
it shares with the monobasic processing site of
prosomatostatin(42, 43) , which is cleaved
inefficiently by PC3(44) . In contrast, transfected AtT-20
cells process prosomatostatin efficiently at this
site(45, 46) . Our results indicate a similar
discrepancy between the apparent lack of cleavage at the monobasic site
in GLP1 by PC3 in vitro versus the high efficiency of tGLP-1
production from proglucagon in AtT-20 and other PC3-expressing cells.
Thus, unless the in vitro activity of PC3 is not
representative of its in vivo activity, this result suggests
that an additional endopeptidase may be required in the regulated
secretory pathway of AtT-20 cells (and intestinal L cells) to produce
bioactive tGLP-1.
Potential candidates for this monobasic cleaving
endoprotease include a putative mammalian homolog to the yeast
YAP3-encoded aspartyl protease (47) . This enzyme has been
shown to efficiently process anglerfish prosomatostatin II at its
monobasic but not at its dibasic site, both in vitro and in vivo(48, 49) . Moreover, an aspartyl
protease has been purified from anglerfish Brockmann bodies, that
specifically cleaves this monobasic site(50) , suggesting that
YAP3 homologs could be present in the endocrine cells of higher
eukaryotes. A second candidate for a GLP-1 convertase is the
dynorphin-converting enzyme, a thiol protease capable of cleaving Dyn
B-29 to Dyn B-13 in vitro (a processing event occurring at a
single arginine). This enzyme is present at high levels in the brain,
the pituitary, and the ileum, and might also be involved in monobasic
processing of precursors other than prodynorphin(51) . Finally,
PACE4 has been reported recently to cleave prosomatostatin at its
monobasic site in the constitutive secretory pathway(44) ,
raising the possibility that an isoform or homolog of this enzyme could
have the same specificity in the regulated pathway, where the monobasic
cleavage of proglucagon is thought to occur. Interestingly, four
isoforms of PACE4 produced by alternative splicing of the mRNA have so
far been cloned(52, 53) . One of these isoforms,
PACE4C, is probably functional and is expressed in the cells, but
not in the
cells of the islets of Langerhans, as shown by
immunocytochemistry(23) . However, it is not known whether this
enzyme enters the regulated secretory pathway.
In conclusion, this
study confirms that PC2 is the convertase responsible for processing
proglucagon to glucagon in the cell and provides evidence that
PC3 is very likely to be the convertase involved in the processing of
proglucagon in L cells. However, the monobasic cleavage necessary to
release the bioactive tGLP-1 is not efficiently performed by PC3 in
vitro, suggesting that an as yet unidentified additional
convertase may also be required to achieve the characteristic pattern
of proglucagon processing of the intestinal L cell. Further
investigation of the susceptibility of the tGLP-1 production to various
protease inhibitors, both in vivo and in vitro in
secretory granule lysates, may lead to a more thorough characterization
of the convertase involved in this monobasic cleavage.