©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Endoproteolytic Cleavage of Its Propeptide Is a Prerequisite for Efficient Transport of Furin Out of the Endoplasmic Reticulum (*)

(Received for publication, September 30, 1994; and in revised form, November 23, 1994)

John W. M. Creemers (1)(§) Martin Vey(§) (2) Wolfram Schäfer (2) Torik A. Y. Ayoubi (1)(¶) Anton J. M. Roebroek (1) Hans-Dieter Klenk (2) Wolfgang Garten (2) Wim J. M. Van de Ven (1)(**)

From the  (1)Laboratory for Molecular Oncology, Center for Human Genetics, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium and the (2)Institut für Virologie, Philipps-Universität Marburg, Robert-Koch-Strasse 17, 35037 Marburg, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)(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.


MATERIALS AND METHODS

Cells and Viruses

PK(15) cells (pig kidney cells) were grown in Dulbecco's medium supplemented with 10% fetal calf serum. NRK cells (normal rat kidney cells) were cultured in Dulbecco's modified medium supplemented with 5% fetal calf serum, 0.45% glucose, and 100 µg/ml gentamycin. COS-1 cells (African Green Monkey kidney cells) were propagated in Iscoves modified medium, supplemented with 10% fetal calf serum. WR strain of vaccinia virus (V.V.:WT) was propagated in CV-1 cells, and virus stocks were prepared as described previously (Roberts et al., 1993). Recombinant vaccinia virus V.V.:T7, which expresses the T7 RNA polymerase, has been described before (Fuerst et al., 1986).

Site-directed Mutagenesis and Production of Recombinant Vaccinia Virus

Construction of recombinant vaccinia virus encoding hemagglutinin (HA) of influenza virus A/FPV/Rostock/34 (H7N1) has been described previously (Roberts et al., 1993). Site-directed mutagenesis was carried out using commercially available in vitro mutagenesis kits (Amersham-Buchler and Promega), as described previously (Creemers et al., 1993). Primers used in the in vitro mutagenesis experiments and names of the corresponding mutants are listed in Table 1.



Metabolic Labeling and Immunoprecipitation Analysis

NRK cells grown to confluence in 60-mm diameter culture dishes were infected with bovine furin recombinant vaccinia virus at a multiplicity of infection of 10. 3.5 h after infection, cells were incubated in methionine-free Dulbecco's modified Eagle's medium for 1 h. Thereafter, cells were grown in the presence of [S]methionine (Amersham-Buchler; 100 µCi/ml; specific activity of 1000 Ci/mmol), as indicated in the legends to Fig. 1and 2. In the case of coexpression experiments, plates of confluent NRK cells in 60-mm dishes were infected with recombinant vaccinia virus expressing fowl plaque virus HA (multiplicity of infection of 5) together with either wild-type vaccinia virus or recombinant vaccinia virus expressing bovine furin (multiplicity of infection of 5). 20 h after infection, cells were first grown in methionine-free medium for 1 h, then labeled for 1 h with [S]methionine (100 µCi/ml), and subsequently, a chase was performed for 1 h.


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. (^2)Coexpression experiments of furin with pro-vWF or HA were performed as described before (Stieneke-Gröber et al., 1992; Creemers et al., 1993).

Endoglycosidase H and -F Digestions

Immune complexes bound to protein A-Sepharose beads were incubated at 95 °C for 5 min in 30 µl of phosphate buffer (50 mM, pH 7.0) containing 0.5% beta-mercaptoethanol and 0.1% SDS. The supernatants were then divided into three aliquots. One served as control. The other two were incubated with either 1 milliunits of endoglycosidase H (endo H) or 1 milliunit of endoglycosidase F/N-glycanase F (endo F) (Boehringer Mannheim). After digestion (37 °C for 12 h), samples were analyzed by SDS-polyacrylamide gel electrophoresis.

Immunofluorescence Analysis

Cells grown on glass slides (10 cm^2) were either incubated 2 h before fixation with 100 µg/ml cycloheximide or remained untreated, as indicated in the legends of Fig. 5and 6. COS-1 cells were fixed 48 h after transfection and NRK cells, 7 h after infection. Cells were then rinsed twice in phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde and 0.2% glutaraldehyde for 15 min. Following fixation, the slides were washed twice in PBS and permeabilized for 30 min with triethanolamine solution (100 mM, pH 8), containing the detergents Triton X-100 (0.5%), SDS (0.5%) and N-lauroylsarcosine (0.5%). The slides were then incubated with blocking buffer (Tris 100 mM, pH 7.4, NaCl 150 mM, 0.2% Triton X-100, block reagent 0.5%, Boehringer Mannheim Biochemica) for 1 h and subsequently incubated for 2 h with mouse anti-furin antibodies (MON-148 and MON-152) diluted in blocking buffer. After extensive washing in PBS-T (PBS containing 0.2% Triton X-100), slides were incubated for 1 h with fluorescein-conjugated rabbit anti-mouse IgG diluted in blocking buffer. Excess antibody was removed by extensive washing in PBS-T. Cells were covered with antifade (Citifluor), coverslipped, and examined with a Zeiss Axiophot microscope equipped with UV optics.


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.




RESULTS

Biosynthesis and Glycosylation of Human and Bovine Furin

Autoproteolytic cleavage of the propeptide from profurin has been suggested to be required for exit from the endoplasmic reticulum and to be necessary but not sufficient for activation of the zymogenic form of furin (Molloy et al., 1994). To obtain more insight in this matter, we have studied zymogen maturation and intracellular distribution of FUR-encoded proteins using a series of human and bovine mutants of furin, including autoproteolytic cleavage site mutants. To allow proper evaluation of the results of such studies, it was of importance to establish first the nature and origin of intermediary products that can be observed during profurin biosynthesis and subsequent maturation. Therefore, we compared biosynthesis and post-translational modification of human and bovine furin in NRK cells using recombinant vaccinia viruses expressing human or bovine furin (Fig. 1). Analysis of pulse-labeled (50 min) wild-type human furin revealed the biosynthesis of two bands with molecular masses of 100 and 94 kDa. After endo H and endo F treatment of the same sample, these bands were found to be shifted toward 94 and 88 kDa, respectively. After a chase of 1 h, a broad band of approximately 100 kDa could be detected, which was endo H-resistant. Upon removal of N-linked glycosylation by endo F, a major protein of 88 kDa, and a minor band with a slightly higher molecular mass, was detected. From these results we concluded that the primary translational product of human FUR, unglycosylated profurin, is a 94-kDa protein. Upon initial glycosylation, its molecular mass is increased to 100 kDa, which decreases to 94 kDa after propeptide removal and may increase again to 100 kDa by the addition of complex oligosaccharides. Thus, it is important to note that the observed 100 kDa band can be composed of both glycosylated endo H-sensitive profurin and the endo H-resistant processed form and that the molecular mass of both unglycosylated profurin and glycosylated endo H-sensitive processed furin is 94 kDa (summarized in Fig. 2). The identity of the deglycosylated furin band migrating just above the 88-kDa band remains elusive, but it could be the result of another post-translational modification, as will be discussed later. These results were confirmed in similar studies of bovine furin. Analysis of pulse-labeled wild-type bovine furin showed two bands with molecular masses of about 104 and 98 kDa. After endo F treatment of the same sample, these bands were found to be shifted toward 98 and 92 kDa, respectively. Endo H treatment of this sample indicated the presence of an endo H-resistant protein of 104 kDa. After a chase of 1 h, a major band of 104 kDa could be detected, which was endo H-resistant. Upon removal of N-linked glycosylation by endo F, only a protein of 92 kDa was detected. In conclusion, these results establish that human and bovine furin are initially synthesized as endo H-sensitive glycoproteins (core proteins of 94 and 98 kDa, respectively), which by propeptide cleavage are converted into endo H-sensitive forms of 94 and 98 kDa, and later on into endo H-resistant forms of 100 and 104 kDa (summarized in Fig. 2). Although biosynthesis of human and bovine furin in NRK cells was found to be similar, the modification of the oligosaccharides of bovine furin into an endo H-resistant form appeared to occur somewhat faster than in case of human furin. The endo H-resistant forms of processed furin points toward their post-ER localization, while endo H-sensitive forms suggest that transport of these furin forms is not yet beyond the ER compartment.


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 Autoproteolytic Cleavage Site Mutants of Human and Bovine Furin

To obtain insight in possible roles of the propeptide of furin, we have studied biosynthesis and post-translational modifications of mutants of human and bovine furin in which basic residues of the autoproteolytic cleavage site had been replaced by non-basic amino acids. A critical role of these basic residues in the sequence preceding the established cleavage sites is generally assumed. In human furin, this sequence contains the 5 basic residues K-R-R-T-K-R, and in bovine furin, the sequence is K-R-R-A-K-R. The mutants are listed in Table 1. Biosynthesis of these mutants was analyzed by means of immunoprecipitation, and it should be noted that this analysis was performed after pulse labeling of the cells for 2 h and no chase.

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.

Substrate Processing Activity of Autoproteolytic Cleavage Site Mutants of Human and Bovine Furin

Although immunoprecipitation analysis suggested that it is critical for autocatalytic processing of furin that the consensus cleavage sequence R-X-K/R-R precedes the actual cleavage site, it was still possible that small amounts of mature furin were present, but escaped detection. A much more sensitive method to determine the presence of mature furin is to assay its biological activity. Therefore, coexpression studies of furin with HA of fowl plaque virus or the precursor of the von Willebrand factor (pro-vWF) were performed. These glycoproteins have previously been shown to be processed by furin (Stieneke-Gröber et al., 1992; Van de Ven et al., 1990). The results of the proprotein processing experiments are summarized in Table 2. Human mutant R-1Q/R-4Q/R-5Q did not possess detectable substrate processing activity. As all the arginine residues in the consensus cleavage sequence preceding the autoproteolytic cleavage site had been substituted in this mutant, it was predicted that autocatalytic processing of this mutant was blocked completely. The results from both the immunoprecipitation analysis and the enzymatic assay strongly confirm that propeptide cleavage is a critical step in zymogen activation and that only processed furin is capable of substrate processing. When mutants R-4Q, R-4Q/R-5Q, or K-2A/R-5A were coexpressed with a substrate (pro-vWF), complete processing was observed in each case. Thus, despite the fact that no mature mutant furin could be detected by immunoprecipitation analysis, the enzymatic assay clearly demonstrated that proprotein processing activity is present, presumably in the form of trace amounts of mature furin.



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.

N-Linked Glycosylation Is Not a Prerequisite for Autocatalytic Maturation of Profurin and Substrate Processing Capability of Furin

In light of the observed N-glycosylation of furin, the question was raised as to whether it was critical for autocatalytical processing of profurin and/or substrate processing capability of furin. The importance of N-linked glycosylation in protein folding and transport has been demonstrated for other proteins. In these proteins, removal of the glycosylation sites resulted in misfolding and inhibition of transport (Machamer and Rose, 1988; Weitz and Proia et al., 1992). As the three potential sites for N-linked glycosylation appear to be conserved in mammalian furins, we have studied this aspect only in bovine furin. Bovine furin mutant T282N/T335N/S447A was constructed in which all three potential N-linked glycosylation sites were changed as indicated. Analysis of its biosynthesis in pulse-chase experiments (Fig. 4) revealed two major bands, which correspond to unglycosylated precursor (98 kDa) and unglycosylated processed furin (92 kDa). Neither endo H nor endo F treatment appeared to have any effect on this mutant, as expected. Although the efficiency of propeptide cleavage seems to be less efficient as compared to wild-type furin, the presence of the processed form indicates that glycosylation is not essential for autocatalytic processing of profurin. After a chase of 1 h a small increase in the molecular weight of part the processed form was noticed, as observed before in human furin (Fig. 1). Coexpression of this mutant with HA as substrate resulted in complete processing of HA (Table 2). From these experiments it can be concluded that glycosylation is not essential for autocatalytic processing of profurin and substrate processing.


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.



Blocking of Propeptide Cleavage Affects Intracellular Distribution of FUR-encoded Proteins

To determine the effect of blocking of the cleavage of the furin propeptide on the intracellular distribution of furin proteins, we compared the intracellular localization of wild-type furin to that of corresponding mutants of it. First bovine furin was studied. Immunofluorescence studies were performed in NRK cells infected with recombinant vaccinia viruses, and the results are shown in Fig. 5. Wild-type bovine furin (Fig. 5B) was predominantly found in juxtanuclear vesicle-like structures, indicative for mature furin to be localized in the Golgi apparatus, as juxtanuclear staining is typical for proteins which are constituents of the Golgi compartment. Cells expressing mutant R-1Q/R-4Q/R-5Q (Fig. 5C) showed typical ER staining; perinuclear staining and staining of a lace-like network. Analysis of maturation-defective mutant R-1Q (Fig. 5D) revealed some juxtanuclear staining in addition to the ER staining. Analysis of NRK cells expressing the bovine furin mutant with the shifted propeptide cleavage site, mutant R-1Q/A-7R (Fig. 5E), showed a similar staining pattern as cells expressing wild-type bovine furin. Apparently the shifted cleavage site does not affect intracellular localization. Finally, the subcellular distribution of the furin protein encoded by bovine furin mutant T282N/T335N/S447A (without N-linked glycosylation) is also similar to that of wild-type furin (Fig. 5F). N-Linked glycosylation, therefore, does not seem to play a determinating role in targeting furin to the Golgi. In conclusion, the results with the maturation-defective furin mutants point toward retention of profurin in the ER.

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.


DISCUSSION

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.


FOOTNOTES

*
This work was supported in part by the Inter-University Network for Fundamental Research sponsored by the Belgian Government (1991-1995), the Geconcerteerde Onderzoekacties 1992-1996, by European Community Contract BIOT-CT91-0302, by Stichting Technische Wetenschappen Contract NCH22.2726, the Deutsche Forschungsgemeinschaft Grant SFB 286, and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this publication and should therefore both be considered first author.

Holder of a fellowship of the Flemish Institute for the Advancement of Science and Technology in the Industry (IWT).

**
To whom correspondence should be addressed. Tel.: +32-16345987; Fax: +32-16-346073; vandeven%molonc%cme{at}cc3.kuleuven.ac.be.

(^1)
The abbreviations used are: vWF, von Willebrand factor; ER, endoplasmic reticulum; HA, hemagglutinin; endo H, endoglycosidase H; endo F, endoglycosidase F; PBS, phosphate-buffered saline.

(^2)
W. Schäfer, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank R. Siezen for helpful suggestions and D. Huylebroeck, P. Groot Kormelink, and E. W. Beek for their contributions to the vaccinia virus experiments. We are also grateful to S. Berghöfer for expert technical assistance, to P. C. Roberts for providing the fowl plague virus hemagglutinin recombinant vaccinia virus, and to G. T. Krause, Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, for synthesizing the bovine furin specific oligonucleotides.


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