(Received for publication, August 30, 1994; and in revised form, October 24, 1994)
From the
Pro-protein and pro-hormone convertases are subtilisin/kexin-like enzymes implicated in the activation of numerous precursors by cleavage at sites mostly composed of pairs of basic amino acids. Six members of this family of enzymes have been identified in mammals and named furin (also called PACE), PC1 (also called PC3), PC2, PACE4, PC4, and PC5 (also called PC6). Multiple transcripts are produced for all the mammalian convertases, but only in the cases of PC4, PACE4, and PC5 does differential splicing result in the modification of the C-terminal sequence of these enzymes. A similar molecular diversity is also observed for the convertases of Hydra vulgaris, Caenorhabditis elegans, and Drosophila melanogaster. In the third species, two genes homologous to human furin called Dfur1 and Dfur2 have been identified. The Dfur1 gene undergoes differential splicing to generate three type I membrane-bound proteins called dfurin1, dfurin1-CRR, and dfurin1-X, which differ only in their C-terminal sequence. By using recombinant vaccinia viruses that express each of the dfurin proteins, we investigated the potential effect of the C-terminal domain on their catalytic specificities. For this purpose, these enzymes were coexpressed with the precursors pro-7B2, pro-opiomelanocortin, and pro-dynorphin in a number of cell lines, and the processed products obtained were characterized. Our studies demonstrate that these proteases display cleavage specificities similar to that of mammalian furin but not to that of PC2. In contrast, we noted significant differences in the biosynthetic fates of these convertases. All dfurins undergo rapid removal of their transmembrane domain within the endoplasmic reticulum, resulting in the release of several truncated soluble forms. However, in the media of cells containing secretory granules, such as GH4C1 and AtT-20, dfurin1-CRR and dfurin2 predominate over dfurin1, whereas dfurin1-X is never detected. While pro-segment removal occurs predominantly in the trans-Golgi network for all the dfurins, in the presence of brefeldin A, only dfurin1-CRR and dfurin2 can undergo partial zymogen cleavage. The conclusions drawn from the results of this study may well be applicable to the mammalian convertases PC4, PACE4, and PC5, which also display C-terminal sequence heterogeneity.
Post-translational endoproteolysis of precursor proteins is one of the mechanisms by which cells increase the diversity of their biologically active products. Initial cleavage of pro-proteins usually occurs at well defined sites consisting generally of pairs of basic amino acids, frequently Lys-Arg or Arg-Arg, but also at specific monobasic sites usually occupied by a single Arg(1, 2, 3) . Recently, a number of mammalian genes and cDNAs encoding subtilisin-like enzymes have been identified, and these candidate processing enzymes were proposed to be responsible for the cleavage at dibasic and monobasic sites of precursor proteins (for reviews see (3, 4, 5, 6) ). The substrate precursors include those of polypeptide hormones, neuropeptides, growth factors, growth factor receptors, certain plasma proteins, and viral envelope glycoproteins. Human furin, which is encoded by the fur gene, is ubiquitously expressed in all tissues and represents the first identified mammalian member of this family of convertases(7, 8) . The other mammalian convertases are the neural and endocrine-specific PC1 ( (9) and (10) ; also called PC3 in (11) ), PC2(9, 12) , the widely distributed PACE4(13) , PC5 ((14) , also called PC6 in (15) ), and the testis-specific PC4(16, 17) . Several of these mammalian enzymes were found to have counterparts in other species, such as the PC1-like (or PC3-like) protein from Hydra vulgaris (18) and anglerfish(19) , the PC1, PC2, and furin-like cDNAs of Aplysia californica(20, 21) , the PC2 and furin-like structures of Lymnaea stagnalis(22) , the furin-like bli-4 gene product of Caenorhabditis elegans(23) , Xen-14 and Xen-18 of Xenopus laevis(24) , and the furin-like enzymes of Drosophila melanogaster(25, 26, 27, 28) .
The process of alternative splicing with the consequent production
of several mRNA transcripts has been demonstrated for several members
of the pro-protein convertase family, including human (h) ()PACE4(13, 29) , rat (r) and mouse (m)
PC4(16) , mPC5 (also called PC6)(14, 30) , Hydra PC1 (also called PC3)(18) , and Drosophila (d) dfurin1(28) . In D. melanogaster, the fur-like sequences dfurin1 and dfurin2 were reported to
originate from two distinct genes(27, 28) . In
addition, Northern blot analysis of Drosophila embryos
revealed that expression of the Dfur1 gene generated four
different sizes of transcripts encoding three proteins differing in
their C-terminal sequence, which were called dfurin1, dfurin1-CRR, and
dfurin1-X(28) . The predicted structural characteristics of the
dfurin1-related enzymes are shown in Fig. 1, where they are
compared to those of dfurin2, hfurin, mPC5 (also called PC6), and
hPACE4. The dfurin1 isoforms are identical in structure from their N
terminus up to their catalytic region but exhibit different structural
domains in their C-terminal segments. Notably, the dfurin1-CRR isoform
possesses a cysteine-rich domain that is also seen in dfurin2, hfurin,
hPACE4, and mPC5 (also called PC6) (Fig. 1). Conservation of the
Cys-rich motif in these various convertases suggests an important
function for this segment of the molecule. However, complete deletion
of this domain in mfurin did not seem to affect the capacity of this
enzyme to intracellularly cleave coexpressed mutated
(M2R
) mouse pro-renin(31) . In an attempt to
understand the functional importance of the C-terminal diversity of
dfurin1-related enzymes, Roebroek et al.(28) coexpressed each of these convertases with either
pro-von Willebrand factor or pro-
-activin as
substrates. In that study, no significant differences with regard to
the cleavage specificity of dfurin1, dfurin1-CRR, and dfurin1-X were
observed. In contrast, although dfurin2 was able to efficiently process
pro-von Willebrand factor, it was highly limited in its capacity to
cleave pro-
-activin(28) . So far, aside from
dfurin1 and dfurin2, no other convertases have been reported in Drosophila. In addition, the dfurin1 isoforms revealed
non-overlapping tissue distribution during the various stages of
embryonic development (28) and were found to be expressed
within various organs, including the central nervous system and
hindgut(26, 28) . This suggests widespread but
distinct functions and/or cellular localization for each gene product
and its isoforms. However, the endogenous Drosophila substrates are not yet known.
Figure 1: Alignment of Drosophila and mammalian pro-hormone convertases. Legend to protein domains is depicted at the bottom, and the amino acid length of each convertase is given at the right of the picture.
In this study, in order to define
the cleavage selectivity of each convertase and its isoforms, we
compared their catalytic properties to those of the mammalian enzymes
PC1, PC2, and furin by cellular coexpression of vaccinia virus
recombinants of dfurins with selected precursor substrates. This
allowed us to probe whether the C-terminal variable segments of the
dfurin1 isoforms can affect their cellular cleavage selectivity. Our
selection of representative substrates was based on the classification
proposed by Bresnahan et al.(32) , where precursors
are subdivided into three categories: Type I precursors contain the
consensus Arg-Xaa-(Lys/Arg)-Arg sequence at their cleavage site.
These pro-proteins include growth factors and proteins usually
processed within cells expressing furin and possibly PACE4 and/or
PC5(7) . Type II precursors exhibit a pair of basic residues at
the site of cleavage but no Arg residue at the P4 position.
Representative examples include most pro-hormones, such as
pro-opiomelanocortin (POMC), which is found in cells containing
secretory granules and is now known to be processed in vivo by
PC1 and PC2(33) . Finally, type III precursors are cleaved at a
monobasic site usually represented by a single Arg
such as the
C-peptide cleavage site of pro-dynorphin(34) . Aside from
defining the cleavage selectivity of each enzyme, we also characterized
the biosynthetic products of each dfurin and defined their kinetics of
synthesis and post-translational modifications.
Figure 5:
Biosynthesis of Dfur1- and Dfur2-encoded proteins in LoVo and BSC40 cells. LoVo and BSC40
cells were infected with either VV:wt (wt), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were
pulse-labeled with [S]methionine and
immunoprecipitated with rabbit anti-dfurin1 (the first wt lane, dfur1, 1-CRR, and 1-X) or
anti-dfurin2 (the second wt lane and dfur2) antisera
as described under ``Materials and Methods.'' Both cells (A) and media (B) were immunoprecipitated. Molecular
mass markers and the positions of the highest molecular forms detected
of the dfurins are indicated.
Figure 8:
Biosynthesis of Dfur1- and Dfur2-encoded proteins in GH4C1 and AtT-20 cells. GH4C1 and
AtT-20 cells were infected with VV:wt (wt), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). 17 h postinfection,
cells were preincubated in medium lacking methionine and then
pulse-labeled with [S]methionine for 2 h and
immunoprecipitated with rabbit anti-dfurin1 (the first wt lane, dfur1, 1-CRR, 1-X) or
anti-dfurin2 (the second wt lane and dfur2) antisera
as described under ``Materials and Methods.'' Both cells (A) and media (B) were immunoprecipitated. Molecular
mass markers and the positions of the highest forms of dfurins detected
are indicated.
Figure 2:
Analysis of proteolytic processing of
pro-m7B2 by the dfurins. LoVo cells were coinfected with 1 pfu of
VV:pro-m7B2 and 1 pfu of VV:wt (wt), VV:mPC1 (mpc1),
VV:mPC2 (mPC2), VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were pulsed for
15 min with [S]methionine and chased for 45 min
in the presence of excess unlabeled methionine. Media were then
immunoprecipitated and resolved by electrophoresis as described under
``Materials and Methods.'' Molecular mass markers and the
relative positions of pro- and mature m7B2 are
indicated.
Figure 3:
Analysis of proteolytic processing of
mPOMC by the dfurins. GH4C1 cells were coinfected with 1 pfu of
VV:mPOMC and 1 pfu of VV:mPC1 (mPC1), VV:mPC2 (mPC2),
VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X-PVV (1-X-PVV), VV:dfurin1-X-PMJ (1-X-PMJ), or VV:dfurin2 (dfur2). Cells were pulsed
for 2 h with [S]methionine. Media were then
immunoprecipitated with either anti-
-END antibody (AT-1) (A) or anti-ACTH antibody (AT-2) (B) and resolved by
electrophoresis as described under ``Materials and Methods.''
Molecular mass markers and the relative positions of POMC,
-LPH,
and
-END (A), and ACTH, glycosylated ACTH (ACTH*), joining peptide-ACTH intermediary processing product (JP-ACTH*), and
-melanotropin-like peptide (
-MSH) (B) are indicated. Wt/mPOMC negative
control coinfection was also performed and immunoprecipitated with both
anti-
-END antibody (AT-1) and anti-ACTH antibody (AT-2). Only the
anti-ACTH immunoprecipitation is shown
here.
Figure 4: Analysis of proteolytic processing of rat pro-dynorphin by the dfurins. As shown in B, LoVo cells were coinfected with 1 pfu of VV:pro-rdynorphin and 1 pfu of VV:mPOMC (mPOMC), VV:mPC1 (mPC1), VV:hfurin (hfur), VV:dfurin1 (dfur1), VV:dfurin1-CRR (dfur1-CRR), VV:dfurin1-X (dfur1-X), or VV: dfurin2 (dfur2). 17 h postinfection, cells were incubated for 3 h with unsupplemented Ham's F-12 medium. Media were collected and resolved by chromatography on Sephadex G-50, and fractions were assayed by radioimmunoassay for C-peptide immunoreactivity as described under ``Materials and Methods.'' An example of the G-50 elution profile assayed by radioimmunoassay is depicted in A, where fractions of the coinfection medium of VV:pro-rdynorphin and VV:dfurin1-CRR separated on G50 were assayed for C-peptide immunoreactivity. Arrows indicate the elution positions of rat pro-dynorphin (1) and C-peptide (2). In B, quantitation of the percent processing into C-peptide for each rat pro-dynorphin coinfection was determined by dividing the sum of the picograms/fraction immunoreactivity found in the C-peptide peak by the total immunoreactivity due to both pro-dynorphin and C-peptide. These percentages were then divided by the mPC1 percent of cleavage value, yielding relative cleavage efficiency values for the dfurin convertases with respect to mPC1.
In order to further define some of the protein
forms observed in LoVo cells, we undertook pulse-chase experiments
using short pulse periods to determine potential precursor-product
relationships for dfurin1- and dfurin1-CRR-immunoprecipitated proteins (Fig. 6). As shown in Fig. 6A, after a pulse of
either 1 or 8 min, we saw the formation of at least two forms of
dfurin1, migrating with apparent molecular masses of 123 and 119 kDa.
As shown in the upper panel of Fig. 6A, the
immunoprecipitation of the dfurin-1 products is specific, because no
bands were observed when we used a normal rabbit serum. Also, after
only 1 min of pulse, we saw the formation of both the 123- and the
119-kDa forms, with the former disappearing within 30 min of chase. In
the bottom panel, this 123 kDa form, which is absent after a 1-h chase,
could represent the precursor of dfurin1 still containing a
transmembrane domain (TMD), because it is never detected in the medium
(data not shown). Similarly, the 119-kDa form would represent the
precursor form lacking its TMD, because it is secreted into the medium
(see Fig. 5B). Furthermore, the difference of 4 kDa
observed between the 123- and 119-kDa forms cannot be due to an
N-terminal truncation of the pro-segment, because we should have
expected a variance of about 17 kDa between the calculated masses of
pro-dfurin1 (88 kDa) and dfurin1 (71 kDa), assuming about 1.5
kDa/N-glycosylation site(25) . We note the slow
migration of the putative TMD-containing pro-dfurin1, which travels
with an apparent molecular mass overestimated by about 35 kDa (123 versus 88 kDa). Such abnormal migration on SDS-PAGE has
previously been observed with a number of proteins, including human
pro-furin and furin, which migrate with apparent molecular masses about
20 kDa higher than expected from their amino acid
sequence(8, 49) . The microsequence of 2
10
cpm of the 119-kDa form of dfurin1 (in LoVo cells)
labeled in [
S]methionine did not reveal the
presence of methionine residues within the first 20 cycles, in
agreement with its tentative assignment as pro-dfurin1 (starting at
residue 152). However, microsequencing of the protein equivalent to the
95-kDa form of dfurin1 (obtained from AtT20 cells; see Fig. 8)
revealed methionines at positions 5, 15, and 17 in agreement with a
protein sequence starting at residue 310 of dfurin1. This result
demonstrates that the smallest dfurin1 form detected represents the
mature enzyme obtained following the removal of its TMD and cleavage of
the pro-segment at the Arg-Ser-Lys-Arg
site.
Figure 6:
A, pulse-chase analysis of dfurin1 and dfurin1-CRR. In the top panel, LoVo cells
were infected with VV:dfurin1 (all lanes), and some cells were
pulse-labeled with [S]methionine for 1 min (p1, p1 c10, and p1 c30) and then chased for
10 (p1 c10) or 30 min (p1 c30). Other cells were
pulsed for 8 min (p8 adf1 and p8 aNRS). In p1, p1 c10, p1
c30, and p8 adf1, cells were
immunoprecipitated with rabbit anti-dfurin1 antiserum, whereas in p8 aNRS, VV:dfurin1-infected cells were
immunoprecipitated with normal rabbit serum. In the bottom
panel, LoVo cells were infected with VV:wt (wt),
VV:dfurin1 (dfurin1), or VV:dfurin1-CRR (dfurin1-CRR). Cells were pulse-labeled with
[
S]methionine for 8 min. Some cells were chased
for 1 (p 8 min. c 1h) or 2 h (p 8 min. c 2h). All
cells were then immunoprecipitated with rabbit anti-dfurin1 antiserum
as described under ``Materials and Methods.'' Molecular mass
markers are indicated. B, biosynthesis of dfurins in the
presence of tunicamycin. BSC40 cells were infected with VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were
pulse-labeled with [
S]methionine in the presence
of tunicamycin for 1 h and immunoprecipitated with rabbit anti-dfurin1
antiserum (dfur1, 1-CRR, and 1-X) or rabbit
anti-dfurin2 antiserum (dfur2) as described under
``Materials and Methods.'' Molecular mass markers are
indicated. C, biosynthesis of dfurins in the presence of
ionophore A23187. BSC40 cells were infected with VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), VV:dfurin1-X (1-X), or VV:dfurin2 (dfur2). Cells were
pulse-labeled with [
S]methionine in a
calcium-free medium containing A23187 for 1 h and immunoprecipitated
with rabbit anti-dfurin1 antiserum (dfur1, 1-CRR, and 1-X) or rabbit anti-dfurin2 antiserum (dfur2) as
described under ``Materials and Methods.'' Molecular mass
markers are indicated.
When dfurin1-CRR was pulse-labeled for 8 min followed by a chase of 1 and 2 h (Fig. 6A), we observed the formation of a major 153-kDa form and the transient production of a minor 162-kDa protein that disappears after a chase of 1 h. Using similar arguments to those used for dfurin1, the 162-kDa form likely represents the pro-dfurin1-CRR with its TMD, whereas pro-dfurin1-CRR lacking the TMD may be represented by the 153-kDa form. Here also, the 11-kDa difference in masses between these forms (average of four separate experiments) is smaller than that expected from the loss of the N-terminal pro-segment, for which a shift of about 17 kDa would be expected (113 and 96 kDa for pro-dfurin1-CRR and dfurin1-CRR, respectively)(28) .
As shown in Fig. 6B, treatment of VV:dfurin-infected BSC40 cells with tunicamycin reveals that all dfurins are N-glycosylated. The apparent molecular masses of the major forms are 105, 139, 145, and 178 kDa for dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2, respectively. Accordingly, in the presence (Fig. 6B) and the absence (Fig. 5A) of tunicamycin, the observed molecular masses of the major intracellular forms differ by about 5, 8, 10, and 13 kDa for dfurin1, dfurin1-CRR, dfurin1-X, and dfurin2, respectively. It is interesting to note that in the presence of tunicamycin, we also detected small amounts of larger molecular forms migrating at 118, 150, and 157 kDa for dfurin1, dfurin1-CRR, and dfurin1-X, respectively. These may represent the pro-forms still containing the TMD. In addition, we note that prevention of N-glycosylation causes the appearance of excessive degradation products, as was originally reported for PC1 and PC2, for which such degradation was shown to occur within the endoplasmic reticulum (ER)(39) .
Finally as shown in Fig. 6C, in BSC40 cells infected with VV:dfurins in the
presence of the Ca ionophore A23187, only the
putative pro-dfurins lacking the TMD are detectable by
immunoprecipitation, because the molecular masses of the major forms
are virtually the same as those seen in a similar 1-h pulse in the
absence of this Ca
-depleting agent (compare Fig. 5A and Fig. 6C). Because we could
not detect the forms that presumably lack their pro-segment in the
presence of A23187, this suggests that the cleavage of the pro-sequence
is largely inhibited under conditions of low Ca
concentrations. In contrast, because we did not observe the
higher molecular mass forms still containing the TMD, this implies that
A23187 does not significantly affect the removal of this C-terminal
domain.
In order to define whether the shorter forms of the dfurins
are produced within the ER/Golgi stacks, we repeated the pulse labeling
of LoVo cells infected with the various vaccinia virus recombinants of
dfurins both at the permissive 37 °C and restrictive 20 °C
temperature, as well as in the presence of the fungal metabolite
brefeldin A. It is well known that at 20 °C, the transport of
membrane glycoproteins from the trans-Golgi network (TGN) to
the cell surface is severely retarded(50) , whereas brefeldin A
causes the redistribution of the cis- and medial Golgi stacks
to the ER(51) , thus preventing traffic from this compartment
to the TGN. The data in Fig. 7A demonstrates that after
a 2-h pulse with [S]methionine performed at 37
or 20 °C, the processing pattern of the dfurin1 isoforms and
dfurin2 is very similar at both temperatures. No immunoreactive
dfurin1- or dfurin2-related proteins are detected in the medium at 20
°C, confirming that secretion is blocked at this temperature (data
not shown). This suggests that all observed processing events occur
while the proteins transit from the ER up to the TGN and not during or
after secretion. However, in the presence of brefeldin A (Fig. 7B), the protein forms detected are mostly the
high molecular mass pro-forms lacking the TMD with small amounts of
shorter forms of dfurin1-CRR and dfurin2 detectable, suggesting that
the removal of the pro-segment of the dfurins occurs in the TGN but can
also happen earlier for dfurin1-CRR and dfurin2.
Figure 7:
A, comparative biosynthesis of Dfur1- and Dfur2-encoded proteins at 37 and 20
°C. LoVo cells were infected with VV:hfurin (hfur),
VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), or
VV:dfurin2 (dfur2). Cells were pulse-labeled with
[S]methionine for 2 h at either 37 or 20 °C
as indicated and immunoprecipitated with anti-hfurin (hfur),
anti-dfurin1 (dfur1 and 1-CRR), or anti-dfurin2 (dfur2) antisera as described under ``Materials and
Methods.'' Molecular mass markers are indicated. B,
biosynthesis of dfurin1, dfurin1-CRR, and dfurin2 in the presence of brefeldin A. LoVo cells were infected with
VV:dfurin1 (dfur1), VV:dfurin1-CRR (1-CRR), or
VV:dfurin2 (dfur2). Cells were pulse-labeled with
[
S]methionine in the presence of brefeldin A for
1 h and immunoprecipitated with anti-dfurin1 (dfur1 and 1-CRR) or anti-dfurin2 (dfur2) antisera as described
under ``Materials and Methods.'' Both cells and media were immunoprecipitated. Molecular mass markers are
indicated.
Although it has been established that multiple mRNA forms
exist for each one of the six known mammalian convertases belonging to
the kexin/subtilisin
family(3, 4, 5, 6, 52, 53) ,
so far only the predicted protein sequences of
PACE4(13, 29) , PC4(16, 54) , and PC5
(also called PC6) (30) have been reported to be affected by
this diversity. The physiological advantage provided by this C-terminal
diversity is not yet known, especially because the complete deletion of
the C-terminal Cys-rich segments of mfurin (31) does not seem
to affect the intracellular cleavage capability of this enzyme in the
TGN. Conceivably, the different C-terminal sequences present in the
various isoforms may impart a specific cellular address, as is the case
for the amidation enzyme isoforms(55) . Alternatively, the rate
of processing of potential substrates or the cleavage selectivity of
the various isoforms for different precursors may be affected by their
C-terminal diversity. The availability of three different dfurin1
isoforms (28) allowed us to address this question in the
context of mammalian cells. The identification of a second furin-like
enzyme in Drosophila, dfurin2(27) , gave us the
opportunity to compare the cleavage selectivity of dfurin1 and dfurin2
convertases with three types of precursors, each exhibiting a different
processing motif. Our results using pro-7B2 and those of Roebroek et al.(28) using pro-von Willebrand factor, both of
which contain the consensus cleavage motif
Arg-Xaa-(Lys/Arg)-Arg
, show
that all dfurins are able to process type I precursors. Our data extend
the characterization of the dfurins to show that these convertases
exhibit cleavage selectivities closer to those of mammalian furin or
PC1, but not PC2, toward either type II (POMC) or type III
(pro-dynorphin) precursors. Although quantitative differences in the
amounts of generated products were noted between each dfurin1 isoform,
we cannot exclude the fact that some of these variations are not due to
the expression of different levels of these enzymes, especially in the
case of dfurin1-X. Indeed, as shown in Fig. 3and Fig. 8as well as in the microsequencing results, it appears that
the degree of processing of POMC can be directly correlated with the
levels of immunoprecipitated convertases in which the pro-segment has
been excised. In addition, coexpression studies with pro-7B2 (Fig. 2) and pro-dynorphin (Fig. 4) show that dfurin1 and
dfurin1-CRR are closer to mammalian furin rather than to PC1 in their
substrate cleavage efficacy. Our data with pro-7B2 (Fig. 2) and
POMC (Fig. 3) demonstrated that dfurin2 exhibits similar
cleavage efficiency and selectivity to dfurin1 and dfurin1-CRR. In
contrast, the extent of cleavage of pro-dynorphin by dfurin2 is similar
to that of dfurin1-X and about 40% of that observed for dfurin1 and
dfurin1-CRR (Fig. 4). This difference in cleavage efficiency of
dfurin1-X and dfurin2 toward rat pro-dynorphin compared with pro-m7B2
is substrate-determined and not cell typedetermined, because both
studies were conducted in LoVo cells. A similar lower efficiency of
cleavage of pro-
-activin by dfurin1-X as compared with
the two other dfurin1 isoforms was reported earlier(28) ,
whereas dfurin2 did not cleave this substrate but processed pro-von
Willebrand factor(28) . (
)Thus, the C-terminal
domain of the dfurin1 isoforms does not affect their cleavage
selectivity. In no case did these convertases exhibit a PC2-like
processing pattern. It is therefore unlikely that the protein diversity
generated by the differential splicing of the Dfur1 gene can
generate an enzyme with a similar cleavage preference as that of PC2,
suggesting that the true Drosophila PC2-like convertase has
yet to be identified. In this regard, a PC2-like convertase was
recently cloned from C. elegans, an organism more
evolutionarily ancient than D. melanogaster(56) .
Because the C-terminal variability of the dfurin1 proteins does not
affect their cleavage specificity, is it possible that this domain
influences the biosynthetic transformations undergone by the dfurins?
To answer this question, we examined the fate of these proteins in
several cell types. Originally, Roebroek et al.(28) reported two forms of dfurin1 of approximate molecular
masses of 115 and 110 kDa in COS cells transfected with a dfurin1 cDNA.
In the two constitutive cells used in this vaccinia virus infection
study, LoVo and BSC40, using the same antibodies as Roebroek et al.(28) , we were able to detect up to three forms of dfurin1
with molecular masses of 123 (Fig. 6A), 119 and 95 kDa (Fig. 5B), with the longest form only observed when
short pulse periods were used (Fig. 6A). It is likely
that the two shorter forms are equivalent to those reported in the COS
cells study (28) . Because our data show that these smaller two
forms are secreted into the medium and that microsequencing confirmed
their assignment as pro-dfurin1 and dfurin1, the 123-kDa form should
represent pro-dfurin1 with its TMD, a form that rapidly disappears
during the chase (Fig. 6A). The proposed cleavage of
the 123-kDa form of dfurin1 generating the soluble smaller proteins and
occurring N-terminal to the TMD seems to be unaffected by the presence
of either the Ca ionophore A23187 (Fig. 6C) or brefeldin A (Fig. 7B).
This suggests that the C-terminal TMD cleavage occurs early along the
biosynthetic pathway, most probably within the ER. Because treatment
with A23187 inhibits the formation of dfurin1, the Ca
dependence of the TMD cleavage reaction is not the same as that
of the pro-dfurin1 to dfurin1 processing. The same conclusion was drawn
for the other dfurins.
Similar to the results with dfurin1, our
results with dfurin1-CRR demonstrate the production of a transient
cellular 162-kDa form (Fig. 6A), which we believe
represents pro-dfurin1-CRR with its TMD, whereas the other soluble
forms represent pro-dfurin1-CRR, dfurin1-CRR, and a shorter
C-terminally truncated form, respectively, all lacking the TMD because
they can be detected extracellularly. Both the dfurin1-X and dfurin2
proteins that immunoprecipitated were likewise found in the media of
infected cells. This implies that the TMD of all dfurins is rapidly
removed, yielding a pro-dfurin form that is then further processed to
dfurin. Only in the case of dfurin1-CRR did we observe a third smaller
form in both cells and media. Although the processing sites that result
in TMD cleavage of the dfurins are difficult to define in view of their
abnormal migration on SDS-PAGE, we observed that this shortening of the
dfurins is not Ca-dependent and can occur in LoVo
cells, which are devoid of endogenous hfurin activity(37) .
When hfurin and mPC6-B (30) are overexpressed in cell lines,
soluble forms are also observed arising from the partial loss of their
TMD. However, this shedding event occurs after the exit of hfurin and
mPC6-B from the ER, possibly within the TGN or at the cell
surface(49, 57) . (
)
The brefeldin A and temperature block experiments demonstrate that the processing of pro-dfurins to dfurins occurs in the TGN, whereas the zymogen cleavage of mammalian pro-furin to furin has been demonstrated to occur in the ER(49) . Only in the case of dfurin1-CRR and dfurin2 did we observe some processing in the presence of brefeldin A (Fig. 7B). Could it be that the furin-like convertases endowed with Cys-rich motifs can undergo an earlier pro-segment removal, as is the case with dfurin1-CRR, dfurin2, and mammalian furin, whereas those lacking this structural characteristic, such as dfurin1, dfurin1-X, and PACE4C, undergo later processing? Future work on the definition of the functional role of the Cys-rich motif in convertases will undoubtedly shed more light on this question.
In regulated cells, such as AtT-20 and GH4C1, we observed an intracellular pattern of immunoprecipitated proteins (Fig. 8A) similar to that seen in the constitutive cells. In contrast, in the media of AtT-20 and GH4C1 cells, the smaller forms of dfurins are predominantly secreted (Fig. 8B), especially for dfurin1-CRR and dfurin2(26, 27, 28) . Thus, the presence of the Cys-rich motif may not only confer to dfurin-CRR and dfurin2 the ability to undergo earlier zymogen processing along the secretory pathway but also an increased probability of C-terminal cleavage and secretion in regulated cells.
In conclusion, the data presented in
this work showed that the three isoforms of dfurin1 and dfurin2 have
similar characteristics to mammalian furin in terms of both catalytic
activity and loss of their TMD. The differences in the C-terminal
structure of the dfurin1 isoforms do not seem to influence their
catalytic activity but may affect the rate and cellular site of
processing of their pro-segment and ultimately influence their
residence in different organelles. It is therefore possible that each
isoform that is expressed in a tissue-specific manner at different
stages of the Drosophila embryonic development (26, 28) may exert actions on specific substrates,
which are coordinately expressed. Some of the possible Drosophila precursor substrates that are cleaved at paired basic residues
include those related to the transforming growth factor- family,
such as the decapentaplegic protein(58) , the integrin
-chain(59) , and insulin-like pro-receptor(60) .
The conclusions drawn from the results of this work may well be
applicable to the mammalian PC5 (also called
PC6)(14, 30) , PACE4(29) , and PC4 (16) , because these proteins also exhibit several isoforms,
some of which contain the same Cys-rich motif as that found in
dfurin1-CRR (26, 28) and dfurin2(27) . The
results obtained from this study should help to broaden our
understanding of the role of differentially spliced forms of
pro-protein convertases both in mammalian and non-mammalian systems,
where such diversity has also been reported (for reviews see Refs. 4,
5, and 52). Future work will undoubtedly lead to a more detailed
definition of the roles of the C-terminal domains of these varied
pro-protein and pro-hormone convertases.