(Received for publication, September 30, 1994; and in revised form, November 23, 1994)
From the
The trans-Golgi network (TGN) proprotein convertase furin is synthesized in a zymogenic form and is activated by intramolecular, autoproteolytic cleavage of the propeptide from its precursor. To obtain insight in possible functions of the furin propeptide, we have studied biosynthesis, propeptide cleavage, biological activity, and intracellular localization of human and bovine furin. Analysis of autocatalytic cleavage site mutants of furin revealed that efficient propeptide cleavage requires the presence of the complete furin cleavage consensus sequence Arg-X-Lys-Arg. In studies of a mutant in which the P1+P4+P5 residues of the autoproteolytic cleavage site were substituted, no substrate processing activity could be demonstrated, indicating a complete block of maturation. In immunofluorescence analysis, this mutant was found in the endoplasmic reticulum (ER), suggesting ER retention of profurin. This ER retention, however, appeared saturable. Furin proteins encoded by oxyanion hole mutant N188A and negative side chain mutant D248L, which possess autoprocessing activity but lack substrate processing activity, were found in the Golgi and the ER, respectively. Finally, analysis of a furin mutant, in which all three potential sites for N-linked glycosylation were altered, revealed autocatalytic cleavage, substrate processing, and transport to the Golgi. Our results indicate that cleavage of the propeptide occurs in the endoplasmic reticulum and is necessary but not sufficient for transport of furin out of this compartment.
An important step in the bioactivation of many secretory proteins in eukaryotes is post-translational endoproteolysis of inactive precursor proteins at sites of paired or multiple basic residues (Douglass et al., 1984; Sossin et al., 1989). This activation step is mediated by enzymes of a novel family of subtilisin-like serine endoproteases, of which kexin, encoded by the KEX2 gene of Saccharomyces cerevisiae, was discovered first (Julius et al., 1984). The mammalian prototype is furin, which is encoded by the FUR gene (Roebroek et al., 1986; Van den Ouweland et al., 1989, 1990; Barr et al., 1991). Endoproteolytic cleavage activity of furin and its specificity for basic amino acid motifs, principally Arg-X-Lys/Arg-Arg, was established in coexpression experiments (Van de Ven et al., 1990; Bresnahan et al., 1990; Wise et al., 1990). Based on the conserved structure of their active centers, other members of this novel family of eukaryotic subtilisin-like serine proteases have been identified (for reviews: Barr, 1991; Van de Ven et al., 1992; Halban and Irminger, 1994).
Structurally, furin is highly similar to kexin (Van de Ven et al., 1990). Its subtilisin-like catalytic domain is
preceded by a cleavable signal peptide and a prodomain. The
catalytically essential residues are the catalytic triad consisting of
His, Ser
, Asp
, and the
Asn
of the oxyanion hole (Leduc et al., 1992;
Creemers et al., 1993). Carboxyl-terminal of the catalytic
domain, the so-called ``middle'' domain is located, which
contributes to the enzymatic activity as indicated by deletion mutants
(Hatsuzawa et al., 1992; Creemers et al., 1993). A
cysteine-rich region of yet unknown function precedes the hydrophobic
transmembrane domain which is assumed to anchor the molecule to the
membrane. Furin is ubiquitously expressed (Schalken et al.,
1987) and has been shown in coexpression experiments to cleave a wide
range of precursor proteins; including membrane bound glycoproteins,
like the human insulin receptor precursor (Yoshimasa et al.,
1990), as well as precursors of secreted proteins, like those involved
in blood coagulation such as the von Willebrand factor (vWF) (
)(Van de Ven et al., 1990; Wise et al.,
1990). Many enveloped viruses and intracellular pathogens also make use
of furin expressed in their host cells (Klenk et al., 1993),
like hemagglutinin of fowl plague virus (Stieneke
Gröber et al., 1992), and the envelope
glycoprotein of human immunodeficiency virus (Hallenberger et
al., 1992).
In Western blot analysis of recombinant furin expressed in COS-1 cells, two glycoproteins are detected, one of 100 kDa and the other of 94 kDa (Van de Ven et al., 1990; Creemers et al., 1992). It was assumed that the 100-kDa protein represented profurin, and the 94 kDa a processed form of furin, lacking the propeptide. Amino-terminal sequencing of the 94-kDa furin protein confirmed this prediction (Leduc et al., 1992). Evidence that the 100-kDa protein band contained profurin was provided, using an antibody specific for profurin (Creemers et al., 1992). Via several independent lines of evidence, it was established that proprotein processing activity was associated with the 94-kDa protein and not the 100-kDa furin protein (Leduc et al., 1992; Creemers et al., 1993). Furthermore, activation of profurin has been shown to be an intramolecular autoproteolytic process (Creemers et al., 1993). Pulse-chase experiments of recombinant furin expressed in COS-1 cells revealed that part of profurin was rapidly processed (0-20 min), while the remaining part remained unprocessed even after extended chase periods.
The current knowledge of the function of the prodomain of furin and its maturation pathway is limited. For other proteases, it has been shown that propeptides can play a fundamental role in folding, intracellular transport, and inhibition of enzyme activity (for review see Baker et al., 1993). In an earlier report (Rehemtulla et al., 1992), the propeptide of furin has been shown to be indispensable for maturation and the requirement for a post-ER compartment for autoprocessing was postulated. In a more recent study (Molloy et al., 1994), it was suggested that proregion cleavage is necessary but not sufficient for furin activation, and it was suggested that autoprocessing is an early process taking place in the ER and that propeptide cleavage may be required for exit of furin from this compartment. To obtain further insight in possible roles of the proregion of furin, we have investigated biosynthesis, propeptide cleavage, biological activity, and intracellular localization of a panel of human and bovine furin mutants and compared the results of these studies to those obtained with the wild-type furins of these species.
Figure 1:
Biosynthesis of human and bovine furin.
4.5 h after infection of NRK cells with recombinant vaccinia viruses
encoding wild-type human furin (V.V.:hfur) or wild-type bovine furin
(V.V.:bfur), cells were labeled with
[S]methionine for 50 min and, subsequently,
chased for 0 or 1 h as indicated. Furin proteins were
immunoprecipitated from cell lysates. Thereafter, samples were either
analyzed directly(-) or after incubation with endo H (H)
or endo F (F). Samples were analyzed by SDS-polyacrylamide gel
electrophoresis.
PK(15) cells were
infected/lipofected as described before and labeled 18 h after
infection (Creemers et al., 1993). COS-1 cells were
transfected as described before and labeled 48 h after transfection
(Creemers et al., 1993). After lysis cells were analyzed by
means of immunoprecipitation as described before
(Stieneke-Gröber et al., 1992; Creemers et al., 1993). For analysis of proteins encoded by human FUR sequences, monoclonal antibodies MON-148, and MON-152 were
used (Van Duijnhoven et al., 1992). For analysis of bovine FUR-encoded proteins, a polyclonal antibody was used. ()Coexpression experiments of furin with pro-vWF or HA were
performed as described before (Stieneke-Gröber et al., 1992; Creemers et al., 1993).
Figure 5: Intracellular localization of wild-type bovine furin and corresponding mutants expressed in NRK cells. Subconfluent NRK cells were infected with recombinant vaccinia viruses encoding encoding wild-type bovine furin (V.V.:bfur) (B), or bovine furin mutants R-1Q/R-4Q/R-5Q (V.V.:R-1Q/R-4Q/R-5Q) (C), R-1Q (V.V.:R-1Q) (D), A-7R/R-1Q (V.V.:A-7R/R-1Q) (E), or T282N/T335N/S447A (V.V.:T282N/T335N/S447A) (F). In A, results with non-infected cells are shown. After infection, cells were treated with cycloheximide for 2 h, fixed, and analyzed as described under ``Materials and Methods.'' Scale bar represents 20 µm.
Figure 2:
Maturation pathway of furin. Domains of
furin are indicated as: pro (propeptide), catalytic, middle, CRR (cysteine-rich region), TM (transmembrane
domain), and cyt (cytosolic tail). Amino acid residues of the
catalytic triad (Asp, His
, Ser
)
and oxyanion hole (Asn
) are indicated with one-letter
abbreviations. Open hexagons represent high mannose
oligosaccarides; filled hexagons represent complex
oligosaccharides. Molecular masses and the intracellular localization
of the different intermediates are
indicated.
Biosynthesis and maturation of wild-type human furin and four
cleavage site mutants of it were studied in V.V.:T7-infected PK(15)
cells transfected with FUR constructs expressing furin under
control of a T7 promoter (see ``Materials and Methods''). In
case of wild-type human furin, processing of profurin can be seen, as
demonstrated in Fig. 3A. Some unprocessed profurin (100
kDa band) was still present, as could also be deduced from the presence
of the 94-kDa deglycosylated protein in endo H- and endo F-treated
samples. Part of this precursor band remained unprocessed, even after a
1-h chase period (data not shown). The human furin mutants, R-4Q
(A-K-R-Q-T-K-R), R-1Q/R-4Q/R-5Q
(A-K-Q-Q-T-K-Q-1), R-4Q/R-5Q (A-K-Q-Q-T-K-R
)
and K-2A/R-5A (A-K-A-R-T-A-R
) all showed a major
band of 100 kDa, representing the uncleaved precursor form of furin. A
minor 94 kDa band was always detected with the four furin mutants. They
most likely represent unglycosylated profurin forms of these mutants,
as they did not shift to a lower molecular weight upon endo H or endo F
treatment. We concluded from these results that apparently none of the
propeptides of these four cleavage site mutants of human furin were
cleaved off, suggesting that autocatalytic processing is strongly
impaired or even blocked if any of the basic amino acids in the
consensus cleavage sequence R-X-K/R-R
is replaced.
This conclusion was further supported by results of similar experiments
in COS-1 cells (Fig. 3B) and by results of experiments
with cleavage site mutants of bovine furin using NRK cells infected
with recombinant vaccinia virus expressing these mutants (Fig. 3C). Processing of the precursor of wild-type
bovine furin was apparently incomplete under the conditions used, as
can be deduced from the presence of a weak 98-kDa protein in the endo
H- and endo F-treated lanes. In the case of bovine furin mutant
R-1Q/R-4Q/R-5Q (A-K-Q-Q-A-K-Q
), which is the bovine
counterpart of human furin mutant with cleavage site sequence
A-K-Q-Q-T-K-Q
, biosynthesis of a single 104-kDa
protein was observed, and this was completely converted into a 98-kDa
protein after endo H or endo F treatment, comparable to the pattern of
the human furin mutant. With mutant R-1Q
(A-K-R-R-A-K-Q
), in which only Arg
was substituted, similar results were obtained as with mutant
R-1Q/R-4Q/R-5Q. In mutant R-1Q/A-7R (R-K-R-R-A-K-Q
),
a furin consensus sequence (R-K-R-R) was created at a position three
amino acids proximal to the wild-type cleavage site. This new cleavage
site comprises an Ala at the +1 position and an Lys at the +2
position, both of which have been shown to be rather unfavorable for
substrate processing (Watanabe et al., 1992; Hosaka et
al., 1991). Our results, however, show that autoprocessing of this
mutant occurs, presumably at the newly created site, indicating that
the shift of the propeptide cleavage site has no major adverse effect.
Figure 3: Biosynthesis and autoproteolytic processing of human and bovine furin mutants. Cells were labeled for 2 h, lysed, and immunoprecipitated. Immunoprecipitates were treated with endo H (H) or endo F (F). Corresponding control samples are indicated(-). A, PK(15) cells were labeled 18 h after infection with V.V.:T7 (all lanes) and lipofected with pSelect (mock), wild-type human FUR (pSelecthfur; indicated with wild-type) or human FUR mutants pSelectR-1Q/R-4Q/R-5Q, pSelectR-4Q, pSelectR-4Q/R-5Q, or pSelectK-2A/R-5A, as indicated. B, COS-1 cells were labeled 48 h after transfection with wild-type human FUR (pSVLhfur) or human FUR mutants pSVLR-1Q/R-4Q/R-5Q, pSVLD248L or pSVLN188A as indicated. Non-transfected cells are indicated with mock. C, NRK cells were labeled 4.5 h after infection with wild-type vaccinia virus (V.V.:wt; indicated with mock), vaccinia virus encoding wild-type bovine furin (V.V.:bfur; indicated with wild-type), or bovine furin mutants V.V.:R-1Q/R-4Q/R-5Q, V.V.:R-1Q or V.V.:A-7R/R-1Q, as indicated.
Finally, it should be noted that some experimental variation was introduced by using different kidney cell lines and different expression systems in the experiments described above. One observed variation relates to the efficiency of processing of furin. This appeared to be considerably lower in PK(15) cells using the V.V.:T7 expression system and in COS-1 cells, expressing furin under control of the SV40 promoter, as compared to NRK cells infected with recombinant vaccinia virus expressing human or bovine furin. Another difference is the degree of endo H resistance of carbohydrates, which seems to be by far not as pronounced in the PK(15)/VV:T7 and the COS-1/SV40 promoter system as in vaccinia virus-infected NRK cells (cf.Fig. 3, A-C). A possible explanation for these differences will be discussed later. Despite these differences, biosynthesis of both human and bovine furin appears to be similar in the different expression systems, allowing comparative evaluation of the data obtained in the various systems.
Similar experiments
were performed with the three autocatalytic cleavage site mutants of
bovine furin expressed by recombinant vaccinia viruses in NRK cells and
with HA as substrate (Table 2). Mutant R-1Q/R-4Q/R-5Q did not
possess substrate processing activity, in agreement with the results
obtained with the corresponding human furin mutant. Bovine furin mutant
R-1Q, which showed no detectable autoproteolytic cleavage in
immunoprecipitation analysis, appeared to completely process HA,
indicating that at least trace amounts of biologically active furin
must have been formed. As the arginine residue at position -1
relative to the wild-type cleavage site was replaced by a glutamine
residue, such cleavage might have taken place carboxyl-terminal of the
remaining A-K-R-R sequence. If so, it is clearly highly inefficient.
Mutant R-1Q/A-7R (R-K-R-R-A-K-Q), in which the
cleavage site was shifted three amino acids proximal to the one in
wild-type bovine furin, showed not only autoproteolytic cleavage, but
also substrate cleavage. In conclusion, the results obtained with the
bovine cleavage site mutants are in agreement with those obtained with
human furin and strongly support the idea that all the basic residues
of the cleavage recognition motif (R-X-K/R-R) preceding the
autoproteolytic cleavage site are highly critical and cannot be
replaced without severely impairing autoprocessing.
Figure 4: Biosynthesis and autoproteolytic processing of a bovine furin mutant without N-linked glycosylation (mutant T282N/T335N/S447A). Experiments were performed as in Fig. 1.
Similar immunofluorescence studies were performed in COS-1 cells using wild-type human furin and mutant R-1Q/R-4Q/R-5Q (Fig. 6). The results of these studies support the observations obtained with bovine furin described above. We also investigated the intracellular localization of furin mutants N188A and D248L. For both mutants, it has previously been shown that although propeptide cleavage occurs normally, they do not possess substrate processing activity (Creemers et al., 1993; see also Fig. 3B and Table 2). When wild-type human furin (Fig. 6A) and mutant N188A (Fig. 6C) were expressed in COS-1 cells under control of a SV40 late promoter, strong Golgi staining was observed. However, a remarkable difference was noticed for the mutants R-1Q/R-4Q/R-5Q (Fig. 6B) and D248L (Fig. 6D). Both mutants showed both ER and Golgi staining, indicating only partial retention of furin in the ER. The ratio ER/Golgi staining did not change after cycloheximide treatment (only untreated cells are shown). Intracellular distribution of human furin was also studied in NRK cells (data not shown). In cells expressing wild-type human furin or mutant N188A, staining was predominantly found in juxtanuclear vesicle-like structures whereas cells expressing mutant R-1Q/R-4Q/R-5Q or D248L showed typical ER staining; a perinuclear ring and staining of a lace-like network were observed. The results obtained with the maturation-defective mutant of human furin points again toward retention of profurin in the ER. The staining pattern observed in cells expressing mutant D248L indicates retention of the processed form of mutant D248L in the ER.
Figure 6: Intracellular localization of wild-type human furin and corresponding mutants expressed in COS-1 cells. 48 h after transfection of subconfluent COS-1 cells with wild-type furin DNA (pSVLhfur) (A) or DNA of furin mutants R-1Q/R-4Q/R-5Q (pSVLR-1Q/R-4Q/R-5Q) (B), N188A (pSVLN188A) (C), or D248L (pSVLD248L) (D), cells were fixed and used for immunofluorescence studies as described under ``Materials and Methods.'' Scale bar in each of the figures represents 10 µm.
In conclusion, the autoproteolytic cleavage site mutants of human and bovine furin that are defective in autoprocessing appear to have a subcellular distribution that is different from wild-type furin as they show staining of an extensive lace-like network and a perinuclear ring compared to the strong juxtanuclear staining of the wild-type furins. Intriguing is the observed difference in subcellular localization of the negative side chain mutant D248L and the oxyanion hole mutant N188A (ER and Golgi, respectively), as both display autoprocessing activity but no detectable substrate processing activity. Finally, the ER retention seemed to be saturable; high levels of expression of mutant profurin (and processed mutant D248L) in COS-1 cells resulted in only partial ER retention.
In this study we have examined the involvement of the propeptide in maturation and intracellular transport of furin. It was found that cleavage of the propeptide is an essential step in the bioactiviation of furin. Recently, it was suggested that this post-translational modification step alone is insufficient for full activation of furin; the requirement for another modification occurring in a post-ER compartment was suggested (Molloy et al., 1994). In this context, it is of interest to note that the deglycosylated processed form of furin apparently slightly increased in molecular weight during chase periods (cf. Fig. 1, upper panel and Fig. 4). Since this is observed only after a 1-h chase period, this is most likely the result of a post-translational modification occurring in a post-ER compartment.
The retention of
profurin in the ER indicates that autoproteolytic activation occurs in
this compartment, in agreement with the conversion of profurin into
furin within 10-15 min after synthesis (Creemers et al.,
1993; Vey et al., 1995). The retention of profurin in the ER
is in conflict with the data published of Rehemtulla and co-workers
(Rehemtulla et al., 1992), who postulated a post-ER
compartment for autoprocessing of furin. However, the results of Molloy
and co-workers (Molloy et al., 1994), who described possible
ER localization of an active site aspartic acid furin mutant, support
our data. In addition, results of inhibition of propeptide cleavage by
Ca removal show that wild-type bovine profurin is not
transport-competent (Vey et al., 1995). The retention of
profurin but not furin suggests the possible involvement of the
propeptide in the retention process. In conflict with this is the
retention of the processed form of mutant D248L. Residue
Asp
, which is substituted for a leucine in mutant D248L,
was predicted to be located at some distance from the catalytic groove,
possibly interacting with 2 arginine residues of furin itself (Creemers et al., 1993; Siezen et al., 1994). This mutant is
the first example of processed furin which is retained in the ER.
Retention of profurin may therefore not be caused solely by the
presence of the prodomain, but structural features or sequences present
in another domain of furin may play a role. Retention of mutant D248L
in the ER because of misfolding seems unlikely because of the observed
autoprocessing activity of this mutant.
The molecular mechanism of retention of profurin remains to be established. Since profurin does not contain a known ER retention signal, it might be bound to a protein which does contain such a signal. The affinity of such a protein could be high for profurin but low for mature furin. So, after propeptide cleavage, furin could become dissociated from this retention protein and be transported to the Golgi compartment. The fact that mutant D248L displays an increased retention in the ER could be an indication that this particular mutation affects the affinity for this postulated protein in such a way that it is not lost even after propeptide cleavage. The putative retention protein should be abundantly present in NRK cells, since even high amounts of profurin (and processed mutant D248L) are efficiently retained in the ER. Transport of part of profurin to the Golgi compartments in COS-1 cells could reflect saturation of this system. This is supported by the almost complete retention of profurin observed in COS-1 cells when expression levels were still relatively low (e.g. early after infection with recombinant vaccinia virus V.V.:R-1Q/R-4Q/R-5Q; data not shown).
The precise implications of the observed retention need to be elucidated, and the possible involvement of an as yet unknown protein in the ER retention of profurin remains to be demonstrated. However, it is tempting to speculate that such a retention protein might be a molecular chaperone essential for the correct folding of profurin. This hypothesis is based on the results obtained in pulse-chase experiments in COS-1 cells (Creemers et al., 1993). In these experiments, it was found that part of the newly synthesized profurin is rapidly converted into furin, while the remaining part is not processed at all, even after extended chase periods. The processed part could represent furin that was retained in the ER until folding was completed and the propeptide removed. On the other hand, the unprocessed part might have been transported to the Golgi by default, due to lack of sufficient retention protein, where it remained unprocessed. This hypothesis implies that efficiency of maturation should be higher in cells with an efficient and high capacity for retention of profurin, e.g. NRK cells infected with recombinant vaccinia viruses. This was indeed confirmed in pulse-chase experiments in NRK cells infected with recombinant vaccinia virus expressing furin. After a 1-h chase period, all newly synthesized furin was processed (Fig. 1). Candidate retention proteins are ER-specific chaperones (reviewed by Gething and Sambrook, 1992). Of special interest is calnexin, a molecular chaperone that selectively associates with newly synthesized and incompletely folded monomeric glycoproteins (Ou et al., 1993). This association is transient with incompletely folded glycoproteins but forms a more stable association with misfolded glycoproteins.