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Human Molecular Genetics Pages 2039-2044  

Evidence for furin-type activity-mediated C-terminal processing of profibrillin-1 and interference in the processing by certain mutations
Introduction
Results
   Recombinant expression and analysis of wild-type C-terminal subdomains of profibrillin-1
   Effects of mutations on the processing of recombinant rF6
Discussion
Materials And Methods
   Construction of the expression vectors
   In vitro mutagenesis of expression vector pCis-rF6
   Transfection of cells and pulse-chase experiments
   Purification and analysis of the recombinant rF8 polypeptide
   Immunofluorescence experiments of rF6 polypeptides
   Immunoprecipitation of rF6 polypeptides
   Quantification of the processing of rF6
Acknowledgements
References

Evidence for furin-type activity-mediated C-terminal processing of profibrillin-1 and interference in the processing by certain mutations

Evidence for furin-type activity-mediated C-terminal processing of profibrillin-1 and interference in the processing by certain mutations

L. Lönnqvist, D. Reinhardt1, L. Sakai1 and L. Peltonen*

Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland and 1Department of Biochemistry and Molecular Biology, Oregon Health Sciences University and Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97201, USA

Received May 9, 1998; Revised and Accepted September 7, 1998

Fibrillin-1 is a major component of the 10 nm microfibrils of the extracellular matrix (ECM). It is synthesized as an ~350 kDa precursor molecule, profibrillin-1, which is proteolytically processed into its biologically active ~320 kDa form. Furin, a calcium-dependent endoprotease of the subtilisin family, which is known to be the processing enzyme for a variety of proproteins, is believed to be responsible for the N-terminal proteolytic cleavage of profibrillin-1. In this article we provide several lines of evidence that the C-terminal trimming of profibrillin-1 also occurs via a furin-type activity. Edman degradation of a small recombinant C-terminal subdomain of fibrillin-1 revealed complete processing of the peptide immediately after the tribasic recognition sequence (R-X-K/R-R) for furin. In vitro expression experiments using another recombinant construct consisting of the C-terminal half of fibrillin-1 indicated that disruption of the putative recognition sequence for furin by site-directed mutagenesis drastically impairs proteolytic processing of the propeptide. In addition, our results suggest that the N-terminal half of fibrillin-1 is necessary for its incorporation into the ECM.

INTRODUCTION

Fibrillin-1 is a large (~320 kDa) glycoprotein and is thought to be the core component of the 10 nm microfibrils of the extracellular matrix (ECM) (1). Fibrillin-1 is synthesized as an ~350 kDa propeptide, profibrillin-1, which is proteolytically processed into a mature ~320 kDa polypeptide (2). Profibrillin-1 contains recognition sequences for furin, in the unique N- and C-terminal domains. These sequences are conserved in the related fibrillin-2 molecule and in fibrillin-1 molecules of other species (3-5). As yet it is not known how the processing of fibrillin-1 affects its molecular functions, but the processing is believed to play an important role in the assembly of fibrillin-1 into microfibrils.

Furin is a calcium-dependent serine endoprotease (6,7). This member of the subtilisin family is known to be a processing protease for a variety of protein precursors, including von Willebrandt factor (8), the insulin proreceptor (9), the transforming growth factor [beta]-1 precursor (10), the hepatocyte growth factor proreceptor (11) and certain viral envelope glycoproteins and hemagglutinins (12). Furin is membrane associated and circulates between the trans-Golgi network and cell surface (13); a secreted form of furin has also been reported (14,15). Furin is expressed in all tissues and cell lines tested (16,17).

At least three disease-causing, naturally occurring mutations that can potentially affect propeptide processing by furin have been reported. Two sisters with extreme insulin-resistant diabetes had an Arg->Ser substitution at the P1 position of the insulin proreceptor (18). Several hemophilia B patients have been reported to have decreased factor IX activity due to the substitution Arg->Glu at position P4 of profactor IX (19,20). In a large family with six individuals suffering from isolated skeletal features of Marfan syndrome (MFS), an Arg->Trp mutation at position P6 of the profibrillin-1 polypeptide was identified (2). Although this mutation did not disrupt the furin recognition consensus sequence, it was shown that in the patient's fibroblast culture medium half of the profibrillin-1 molecules (presumably those transcribed and translated from the mutant allele) were not converted to mature polypeptides.

In this study we provide evidence by peptide sequencing and in vitro mutagenesis experiments that profibrillin-1 is processed within the unique C-terminal end after the tribasic consensus sequence for furin (R-X-K/R-R). The effects of specific mutations, located close to or within the consensus sequence, on the conversion of profibrillin-1 into the mature form are also studied using a recombinant model.

RESULTS

Recombinant expression and analysis of wild-type C-terminal subdomains of profibrillin-1

A construct coding for exons 64-65 of the FBN1 gene (pCis-rF8) was stably expressed in HT1080 cells. The resultant polypeptide (rF8) was secreted into the culture medium in relatively low amounts (~0.1-1 µg/ml/day) as an ~32 kDa polypeptide. Edman degradation of purified rF8 revealed an N-terminal sequence (STXETDASNIEDQSE) starting immediately C-terminal of the furin consensus sequence (R-X-K/R-R) at position 2732, indicating cleavage by furin or a furin-type activity (Fig. 1). The small N-terminal part of rF8 (the 43rd cbEGF-like repeat and 44 amino acid residues of the unique C-terminus) could not be identified in the culture medium.


Figure 1. Recombinant rF8 peptide was analyzed by SDS-PAGE (10%) under reducing conditions. Positions of globular marker proteins are indicated in kDa. The N-terminal sequence of rF8 obtained by Edman degradation is indicated.

To follow the course of conversion from profibrillin-1 into the mature form, we transiently expressed a larger subdomain of FBN1 (pCis-rF6), consisting of exons 36-65, in COS-1 and 293-EBNA cells. This subdomain has previously been successfully expressed stably in HT1080 cells (21). The expressed product (rF6) was identified by immunoprecipitation of metabolically labeled transfected cells and cell culture medium with a monoclonal antibody specific for fibrillin-1 (mAb 69). An ~215 kDa band was rapidly converted into an ~190 kDa band which corresponded to the expected masses of the proform and the `mature' form of the rF6 peptide, respectively (Fig. 2). In 293-EBNA cells conversion occurred rapidly (complete after a 2 h chase), while this process took somewhat longer in COS-1 cells (90% complete after a 4 h chase). The processed `mature' form of rF6 was only detected in the cell culture medium, not in the cell lysate samples. rF6 could not be detected in extracts of the extracellular layer run on SDS-PAGE. This result was confirmed by immunofluorescence in which the peri-/extracellular immunofluorescence pattern of the transfected and untransfected cell cultures did not display any differences when stained with mAb 69 (data not shown).


Figure 2. Immunoprecipitated recombinant rF6 polypeptides from 293-EBNA cell culture medium at the 2 h chase point. Positions of the ~215 kDa unprocessed (unpr) and 190 kDa processed (pr) recombinant peptide are marked on the left. Individual mutations are marked. WT, wild-type polypeptide; 293-EBNA, untransfected 293-EBNA cells. Note the slightly faster migration of G2689X due to its smaller size and the significant inhibition of conversion, especially of the R2728A and R2731K mutants, as compared with the wild-type.

Effects of mutations on the processing of recombinant rF6

The following mutations were introduced into the pCis-rF6 construct by site-directed mutagenesis: point mutations resulting in amino acid changes within the furin consensus sequence (R2728A and R2731K), point mutations close to the consensus sequence (R2726K and S2732T), mutations found in individuals with MFS (R2726W and R2776X) and another premature termination mutation (G2689X) resulting in a shortened polypeptide (Fig. 3). The effect of the mutations on the conversion of the rF6 proform was analyzed in cell culture medium samples using SDS-PAGE and autoradiography (Fig. 2). The relative amounts representing the proform and the `mature' form of each rF6 mutant at different chase times (2-48 h) were quantified using a Bioimaging Analyzer (Table 1).


Figure 3. Schematic representation of the expression constructs. The pCis-rF8 construct, consisting of exons 64 and 65, was used to produce a small C-terminal peptide fragment to demonstrate the processing site by Edman degradation. The pCis-rF6 expression construct used in this study consisted of exons 36-65 of the FBN1 gene. Both rF6 and rF8, preceded by the signal sequence of a basement membrane protein, BM-40, were cloned in the pCis expression plasmid. The putative cleavage site for furin and the area of interest are marked. In the lower portion of the figure is a magnified scheme of the area of interest. The putative furin cleavage site and the furin consensus sequence (bold) are marked. The mutations studied and their location with respect to the putative cleavage site (P-45 to P43) are shown.

Table 1. Ratio of the recombinant rF6 polypeptides present in the unprocessed/processed form in COS-1/293-EBNA cell culture medium at different chase points as determined with a Bioimaging Analyzer (for details see Materials and Methods)
  2 h 4 h 24 h 48 h
293-EBNA
WT 0/100 0/100 0/100 0/100
G2689X - - - -
R2726W 25.2/74.8 0/100 0/100 0/100
R2726K 0/100 0/100 0/100 0/100
R2728A 85.8/14.1 77.9/22.1 54.4/45.6 23.7/73.6
R2731K 80.9/19.1 70.0/30.0 39.2/60.8 7.4/92.6
S2732T 39.8/60.2 0/100 0/100 0/100
R2776X 0/100 0/100 0/100 0/100
COS-1
WT   10.8/89.2 0/100 0/100
G2689X   - - -
R2726W   44.1/55.9 43.0/57.0 5.3/94.7
R2726K   13.9/86.1 0/100 0/100
R2728A   72.1/27.8 72.8/27.2 73.4/26.6
R2731K   64.2/35.8 71.3/28.7 68.0/32.0
S2732T   39.1/60.9 40.0/60.0 0/100
R2776X   n.d. 0/100 0/100
The G2689X values are marked -, indicating that this peptide is not processed at all. This is to be expected since it terminates before the putative conversion site (P-43). n.d., not determinable. The values for R2776X at 4 h of chase in COS-1 cell culture medium could not be determined due to the small migration difference between the unprocessed and the processed form of this particular mutant. A broader band was observed on SDS-PAGE at this time point, suggesting that both forms of R2776X are present. At later time points only one sharp band migrating at the same level as the wild-type peptide was observed, indicating the presence of completely processed R2776X mutant.


Mutations disrupting the furin consensus sequence at positions P4 (R2728A) and P1 (R2731K) significantly inhibited processing of the propeptide into the `mature' form. A mutation at the P-1 position (S2732T) and an MFS mutation at position P6 (R2726W) also inhibited conversion, although to a lesser extent. A conservative substitution of the residue at position P6 (R2726K) had no effect on conversion. Of the two mutations resulting in premature termination of the polypeptide, G2689X was not processed, as expected, since termination of this polypeptide occurs prematurely and N-terminal of the putative processing site. Due to the small migration difference of the proform and the `mature' form of the R2776X mutant, it was not possible to determine the exact unprocessed:processed ratio in COS-1 cells at the 4 h chase point. At later chase times and in 293-EBNA cells as early as 2 h after synthesis, R2776X was similarly processed as the wild-type (Fig. 2 and Table 1). None of the mutants caused significant intracellular retention of the polypeptides (data not shown). Processing of the propeptides generally occurred faster in 293-EBNA cells and the inhibitory effect of the mutations on conversion was smaller in 293-EBNA than in COS-1 cells. As for the wild-type rF6, we could not detect any evidence, either by SDS-PAGE with ECM extracts or by immunofluorescence, that the polypeptides are incorporated into ECM.

DISCUSSION

Both the unique N- and C-terminal regions of profibrillin-1 contain a putative recognition sequence for furin endoprotease. These signals are conserved between species and are also found in the other member of the fibrillin family, fibrillin-2 (3-5). Sequencing of recombinant fibrillin-1 polypeptides has earlier shown that N-terminal cleavage occurs immediately after the consensus site for furin protease (21). Data obtained from cultured fibroblasts of an individual with MFS indirectly suggested that processing at the C-terminus also takes place after the furin recognition sequence. However, direct evidence for this has been lacking. It has been suggested that the conversion of profibrillin-1 is extracellular and calcium dependent, since the processed form of the polypeptide, fibrillin-1, has not been observed intracellularly and EGTA, a calcium chelator, inhibits conversion (2,22).

In this study we produced a small recombinant C-terminal fragment of fibrillin-1 (rF8) to demonstrate that processing in the C-terminal end occurs immediately after the tribasic recognition site for furin at amino acid residue 2731. Edman degradation of rF8 peptide purified from HT1080 cell culture medium yielded a sequence beginning immediately C-terminal of the expected sequence (R-X-K/R-R), indicating cleavage by furin or a furin- type enzyme. This is the first time that C-terminal conversion of fibrillin-1 has been shown directly, by peptide sequencing, to take place at the furin recognition site.

In order to collect additional data that the C-terminal processing is mediated by furin or a furin-type convertase, we systematically investigated the effects of specific amino acid substitutions on C-terminal processing. For this experiment another recombinant subdomain, consisting of the C-terminal half of the FBN1 gene, was expressed transiently in COS-1 and 293-EBNA cells. The resultant wild-type rF6 propeptide (size ~215 kDa) was efficiently cleaved into a `mature' ~190 kDa polypeptide. Already at 2 h after synthesis the wild-type form was completely processed in 293-EBNA cells, while in COS-1 cells the conversion was somewhat slower. This might be due to the higher furin-like enzyme activity in 293-EBNA cells and/or a higher level of expression of the rF6 propeptide in COS-1 cells. Mutations R2728A (P4) and R2731K (P1), which disrupt the furin recognition sequence R-X-K/R-R, had the most drastic effects on proteolytic cleavage. These mutations significantly inhibited conversion of the rF6 propeptide in both cell lines, suggesting that the proteolytic reaction is indeed mediated by furin or a furin-type activity. A mutation at the P-1 position (S2732T) adjacent to the cleavage site clearly inhibited conversion in COS-1 cells, but had little effect on conversion in 293-EBNA cells. R2726W in position P6 is a mutation found in individuals with isolated skeletal features of MFS (2). This mutation has been reported to completely inhibit processing of the mutant profibrillin-1 molecules in fibroblast cultures of individuals harboring this mutation. In our experiments this mutation substantially decreased proteolytic cleavage in COS-1 and had only a slight reducing effect in 293-EBNA cells. It is possible that furin or furin-type activities are expressed at higher levels in 293-EBNA and COS-1 cells as compared with fibroblasts, thus `overwriting' the inhibitory effect of the R2726W mutation. Alternatively, the cell lines used in our study might express other furin-type enzymes mediating this processing that are not expressed in fibroblasts. When the same residue (Arg2726) was conservatively replaced by Lys, no inhibitory effect on conversion of the rF6 propeptide was observed.

Two mutations resulting in premature termination of the polypeptide were also expressed: G2689X, leading to truncation of the entire unique C-terminus immediately after the last calcium-binding EGF-like repeat at position P43; R2776X, an MFS mutation truncating the polypeptide at position P-45 relative to the furin consensus sequence (23). As expected, rF6, harboring the G2689X mutation, was not found to be subject to furin-mediated conversion since it lacks the recognition sequence. When we analyzed the mutant R2776X we were unable to determine the unprocessed:processed ratio at the 4 h chase point in COS-1 cells due to the small migration difference between the proform and the `mature' form on SDS-PAGE. At later chase times this mutant appeared as a single, sharp band of the same electrophoretic mobility as the `mature' wild-type rF6. Therefore, we conclude that this mutant was completely processed. If this is correct, why does this mutation cause MFS in vivo? Possibly the ~32 kDa C-terminal fragment that is cleaved off has as yet unrecognized functions in the biology of fibrillin-1/microfibrils. For example, it could play a role in the supramolecular assembly of microfibrils. Alternatively, the presence of the entire C-terminal domain prior to proteolytic processing might be important for correct folding of this domain. The lack of residues 2776-2871 in the R2776X mutant could alter the oxidation/reduction state of the two cysteine residues that are located N-terminal of the processing site. This in turn could affect intra- and intermolecular disulfide bond formation in the assembly processes.

We could not extract the rF6 polypeptide from the ECM of the transfected cell cultures nor could we observe any evidence in immunofluorescence experiments that rF6 is incorporated into microfibrils. Since experiments using 293-EBNA cells transfected with the full-length FBN1 cDNA have shown that the full-length recombinant polypeptide is incorporated into the ECM (T.Rantamäki, personal communication), our results suggest that the N-terminal half of fibrillin-1 (transcribed from exons 1-35) is necessary for its incorporation into the ECM. The possibility that rF6 is incorporated into the ECM and secondarily degraded, however, cannot be totally ruled out.

MATERIALS AND METHODS

Construction of the expression vectors

The construction of expression vector pCis-rF6 coding for the C-terminal half of fibrillin-1 (amino acid residues 1487-2871) was described in detail previously (21). To assemble a construct coding for the last cbEGF-like motif and the unique C-terminalend of fibrillin-1 (amino acid residues 2648-2871), primersDR16 (5[prime]-ATAGTTTAGCGGCCTAATGAAGCAAAACCTGGAT-3[prime]) and DR17 (5[prime]-CGTAGCTAGCAGACATCAATGAATGTGGC-3[prime]) were used to amplify template HFBN4 (24). The 682 bp NheI-NotI restricted fragment was then subcloned into the pCis [gamma]1 III3-5 vector (25,26). The resulting plasmid was designated pCis-rF8.

In vitro mutagenesis of expression vector pCis-rF6

To create the desired mutations in the expression construct pCis-rF6, in vitro mutagenesis using a commercially available kit (Chameleon; Stratagene) was used in accordance with the manufacturer's instructions. Seven different mutations were created. The sequences for the antisense oligonucleotides that introduced the mutations were as follows: G2689X, 5[prime]-CATGCCCATTCAAGAAACACAGTGCC-3[prime]; R2726W, 5[prime]-CGTTTCCTGCCCCATTTGGGGTAGC-3[prime]; R2726K, 5[prime]-CGTTTCCTGCCCTTTTTGGGGTAGCC-3[prime]; R2728A, 5[prime]-CTTCTCCGTTTCGCGCCCCGTTTGG-3[prime]; R2731K, 5[prime]-GTTTCGTTTGGCTTTTCCGTTTCCTGC-3[prime]; S2732T, 5[prime]-GTTTCGTTTGTGGTTCTCCGTTTCCTG-3[prime]; R2776X, 5[prime]-GAGTTCTAGGATTCAAACCTTGTTACTGA-3[prime]. The antisense selection primer used to delete a unique NotI restriction site was 5[prime]-CTAGAGCGGCCCCGGACCTCGAG-3[prime].

Transfection of cells and pulse-chase experiments

For COS-1 cells, a DEAE-dextran transfection method (27) was used. One day prior to transfection, 400 000 COS-1 cells were plated in 35 mm dishes (Nunc) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. The cells were transfected with 5 µg wild-type or mutant pCis-rF6 construct. Seventy-two hours after transfection the cells were pulse-labeled. First the cells were starved for 1 h in Cys-free DMEM without FCS, then the cells were labeled for 1 h with 100 µCi [35S]cysteine (Amersham) in 500 µl Cys-free DMEM without FCS and thereafter chased in 500 µl DMEM without FCS. After the desired chase times the medium was removed, the cells lysed and the ECM extracted as described earlier (22).

For 293-EBNA cells the SuperFect transfection method was used in accordance with the manufacturer's protocol (Qiagen). One day prior to transfection 800 000 cells were plated similarly to the COS-1 cells described above. The cells were transfected with 2 µg wild-type or mutant pCis-rF6 construct. Forty-eight hours after transfection the 293-EBNA cells were labeled, chased and then harvested as described above for the COS-1 cells.

A stable HT1080 cell line expressing the recombinant rF8 peptide was established as described earlier (28).

Purification and analysis of the recombinant rF8 polypeptide

One liter of serum-free medium containing rF8 was concentrated to ~35 ml by ultrafiltration, dialyzed against 20 mM Tris-HCl, pH 8.6, and passed over a 1 ml anion exchange column (MonoQ; Pharmacia) equilibrated in the same buffer. The bound material was eluted by an NaCl gradient in equilibration buffer (0-320 mM NaCl in 40 ml). The fractions containing rF8 (~170-240 mM NaCl) were pooled and acidified with 0.1% trifluoroacetic acid. The material was then passed over a 1 ml hydrophobic interaction column (PepRPC; Pharmacia) equilibrated with 0.1% trifluoroacetic acid. The bound proteins were eluted with an acetonitrile gradient in equilibration buffer (0-52.5% acetonitrile in 35 ml). Fractions eluting at ~43% acetonitrile were then used to further analyze rF8. SDS-PAGE was performed in accordance with established procedures (29). For Edman degradation, purified rF8 was analyzed using a protein sequencer (Applied Biosystems 475).

Immunofluorescence experiments of rF6 polypeptides

COS-1 and EBNA-293 were transfected as described above. Seventy-two hours after transfection the resulting hyper-confluent cell layers were processed and immunostained with anti-fibrillin-1 mAb 69 as described earlier (30).

Immunoprecipitation of rF6 polypeptides

Immunoprecipitation of radiolabeled polypeptides was carried out with mAb 69 coupled to protein G-Sepharose (Pharmacia). Aliquots of 100 µl of the sample were incubated with the same volume of buffer containing 2% (v/v) NP-40, 100 mM Tris-HCl, pH 7.3, and 5 mM EGTA with or without 2 µl mAb 69 on a rotating table at 4°C. Thereafter, 30 µl protein G-Sepharose were added to the sample and the incubation was continued for a further overnight period. After incubation the samples were washed four times with 500 µl buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% (v/v) NP-40, 5% (w/v) sucrose, 500 mM NaCl, 0.1% (w/v) SDS and 0.5% (w/v) deoxycholate. Thereafter the samples were washed twice with buffer containing 10 mM Tris-HCl, pH 6.8, and 1 mM EDTA.

Quantification of the processing of rF6

The total protein and immunoprecipitation samples were supplemented with Laemmli buffer containing mercaptoethanol (final concentration 10% v/v) and visualized by 7% SDS-PAGE and fluorography as described earlier (22). The dried SDS-PAGE gels were exposed to an imaging plate (BAS-MP 2040S; Fuji) for 1-4 days and the plate was scanned in a Fuji BAS 1500 Bioimaging Analyzer. The signals were measured using the Tina v.2.09 software package (Raytest) on a microcomputer by outlining the bands representing the unprocessed or processed form of the rF6 polypeptide, determining signal/mm2 and reducing background signal/mm2.

ACKNOWLEDGEMENTS

The financial support of the Maud Kuistila Foundation and Shriners Hospital is gratefully acknowledged.

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*To whom correspondence should be addressed. Tel: +358 9 4744 8393; Fax: +358 9 4744 8480; Email: leena.peltonen@ktl.fi

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