Furin-mediated proprotein processing activity: Involvement of negatively charged amino acid residues in the substrate binding region

Biochbnie (I 994) 76. 210-216

© SociEt~ franqaise de biochimie et biologie mol~culaire / Elsevier, Paris

Furin-mediated proprotein process ng activity: Involvement of negatively charged amino aci residues in the substrate binding region AIM Roebroek, JWM Creemers, TAY Ayoubi, WJM Van de Ven Z,aboratory for Molecular Ontology. Center for Human Genetics, University of Leuven. Herestra Tt49. B-3000 Lt.uven, Belgium

(Received 18 February 1994:accepted 16 May 1994)

Smamary - - Furin, which is encoded by the recently discovered FUR gene, appears to be the first known mammalian member of the subtilisin family of serine proteases with cleavage selectivity for paired or multiple basic residues. A consensus cleavage sequence, Arg-X-Lys/Arg-Arg has been proposed. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Homology modelling of the catalytic domain of this protein suggested that negatively charged amilo acid residues near or in the substrate binding region might contribute to the observed specificity for substrate segments with paired and multiple basic amino acid residu~. To investigate this hypothesis, furin mutants were generated in which negatively charged residues, predicted to be located near or m the substrate binding pockets and involved in interactions with basic residues of the substrate, were replaced by neutral residues. Analysis of processing by these furin mutants of wild.type and cleavage mutants of pro-yon Willebrand factor (pro-vWF) revealed that particular negatively charged residues are critical for srecific cleavage activity.

furin enzyme I proprotein processing / site-directed mutagenesis / substrate binding pockets Introduction

At present, it is well documented that a wide variety of secretory proteins mature from high molecular mass precursor proteins by selective endoproteolytic cleavage within the cells [1-7]. Cleavage of precursors of many hormones and neuropeptides secreted vi:~ the regulated pathway occurs at the carboxyl side of Lys-Arg (KR) or Arg-Arg (RR) sequences in secretory granules of endocrine cells or dense core vesicles of neurons. However, precursors of proteins that are secreted via the constitutive pathway seem to be cleaved at the carboxyl side of more complex basic sequences, the consensus sequence of which can be represented as Arg-X-Lys/Arg-Arg (RXK/RR). Although the existence of endopeptidases with such specificity was already predicted many years ago [1, 2], only recently a number of such processing enzymes have been identiffed (for recent reviews, see [8, 9]). The mammalian prototype is furin [10, 11], which is encoded by the ubiquitously expressed FUR gene [12-15]. The cleavage specificity of furin was predicted on the basis of its sequence similarity ~o the prohormone conv~rtase (PC) kexin, a membrane-associated, Caa+-dependent, subtilisin-like serine endoprotease of yeast Saccharomyces cerevisiae with an established clevvage selectivity for paired basic amino acid residues [6, 16].

Other mammalian proprotein processing enzymes are PAC~4, PC I/PC3, PC2, PC4 and PCS/PC6 (.isoforms A and B) [17-26]. Of these, the PACE4 endoprotease is, like furin, ubiquitously expressed [17]. Expression of the enzymes PC I/PC3 and ~_~2 is neuroendocrine specific [18-21], that of P~,G~germ cell-specific [22, 23] and PCS/PC6 is e ~ r ~ s'~e d in subsets of endocrine and non-endocrine ~ l l s [24-26]. Selective endoproteolysis of substral~s by turin at the RXK/RR consensus cleavage sequence was first demonstrated in coexpression experiments using the precursor of yon Willebrand factor (pro-vWF) [10, 27] or [~-nerve growth factor ([3-NGF) [28] as substrate. Furin-mediated processing of a variety of other precursor proteins has been described now, including precursors for factor IX [29], anthrax toxin protective antigen [30, 31] and viral proteins such as hemagglutinin of influenza virus [32] and envelope glycoproteins of the human immunodeffciency virus- 1 (HIV- 1) [33].

The furin protein Furin is synthesized as a precursor, which consists of a 'prepro' domain (contaiaing a cleavable signal peptide), a subtilisin-like catalytic domain, a 'middle' domain, a cysteine-rich region, a transmembrane

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Fig 1. Schematic representation of protein domains in furin. Protein domains are represented as boxes which are marked as indicated. Domains include a 'prepro' domain, subtilisinlike catalytic domain, middle domain, cysteine-rich region (CRR), transmembrane domain (TM) and cytoplasmic domain (cyt). The relative positions of the essential residues (Asp, His, Asn and Ser) of the active site are indicated within the catalytic domain with the corresponding single lever code (D, H, N and S).

region and a cytosolic tail [10, 11] (see fig 1). All other mammalian endoproteases resemble furin in having a 'prepro' domain, a subtilisin-like catalytic domain and a 'middle domain'. Carboxy-terminally of the 'middle' domain however, the domain structure of the various members diverges. Like furin, PACE4 and PC5/PC6 possess a cysteine-rich region, although theirs is much more extended [17, 24-26]. Only isoform PC6B, probably generated by alternative splicing of the PC5/PC6 gene, has a transmembrane region and cytos.,0(lic tail like furin [26]. The function of the cysteine-ri'~h region, which is also found to be conserved in fiJrin-like enzymes of Drosophila melanogaster, Dfurinl-CRR a~ld Dfurin2 [34, 35], is still unknown. PC I/PC3, PC2 and PC4 lack a cysteine-rich region, transmembrane region and c)~osolic tail [1823]. It should be noted that the highest sequence similarity between these endoproteases is found in the subtilisin-like catalytic domain. The turin 'core protein', the minimal part of prepro-furin needed to sustain,its cleavage activity, consists of the 'prepro', the catalytic and the 'middle' domain. Analysis of deletion mutants of furin, in which carboxy-terminal parts were deleted, indicated that sequences carboxy-termihal of the 'middle' domain were dispensable [36, 37]. Similarly, it was previously shown that the 'P-domain' of kexin, which is homologous to the 'middle' domain of furin, is essential for processing activity of kexin [38]. A mutant form of furin that lacked the 'pro' sequence was non-functional, while addition of the 'pro' sequence of the PC2 enzyme did not restore activity [39], indicating that the 'pro' region is essential for furin activation, possibly by guiding folding of furin into an active enzyme configuration. Furin is initially synthesized as a zymogen, pro-furin, and activated to mature furin by removal of its 'pro' region from the catalytic domain at a consensus sequence for furin cleavage (RTKR). This was demonstrated to occur via an autocatalytical and intramolecular processing event [37, 39, 40]. The subtilisin-like catalytic

domain of furin possesses a characteristic catalytic triad, conserved in all other enzymes of the family [41], consisting of the residues Asp 46, His87 and Ser 261, all of which have been documented to be critical for autocatalytic maturation and substrate processing. Mutation of one of these residues blocked furin activity completely [37, 40, 42]. Another characteristic fe~ ~re ot the active site of furin is the oxyanion hole, with Asn~SS as critical residue; this asparagine is conserved in all other enzymes [41], except for PC2, which has an :~spartic aci~',~esidue [ i8]. Analogous to subtilisin, this oxyanion~i)inding site is believed to stabilize an ox)anion ir~!ermediate that is generated during hydrolysis of tl'.~: scissile peptide bond [43]. Also Asn~88 seems to be essential for substrate processing, although it is less critical for the autocatalytical processing event since some maturation is obser-

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Fig 2. Schematic model of the catalytic domain of human furin based upon ribbon drawing of subtilisin. The catalytic triad residues Asp 46, His s7 and Ser261 are situated at the top centre. Predicted positions of inserts relative *.o subtilisin are represented in solid black and pos~ibie positions o( two stabilizing Ca2+ ions are shown as hatched spheres~ Predicted disulphide bridges are shown as dotted li~es. Negatively charged side chain groups or, the subs:'~ate binding face are drawr as forked stalks Most of thes~.~charges are not present ir~ equivalent posmons m subtd~sJ ,s and thermitase. They seem to be located in or near the S1, $2 and $4 binding laockets for lysine and/or arginiae and, therefore, they might interact directly with paired basic residues in the substrate [44]. Reprinted by permission of Kluwer Academic Publishers [ 10]. "'

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The three-dime~.~sional structure of the catalytic Computer assisted homology modelling provided insight in the tl',xee-dimensional s~ructure of the catalytic domain of turin. Based on aniino acid sequence similarity ~t,d the established three-dimensional structures of thq~ prokaryotic serine proteases, thennitase, subtilisin Carlsberg and subtilism BPN', a model of the three-dimensional structure of the catalytic domain of humar, furin was proposed [I0, 411. The three-dimensional structure was predicted to consist essentially of the framework core of the three prokaryotic proteins with several short insertions in external loops and connections between a-helices and 13-sheets (see fig 2). Relevz,nt to note here is that all essential o~helix and 13-sheet secondary s~ructure elements of the extended consensus core of the subtilisins could also be identified in turin. Two potential calcium-ion binding sites and two potential disulphide, bonds were predicted. This homology modelling also suggested appropriate positioning of the residues of the catalytic triad (Asp4~, His87 and Ser2~l) and the oxyanion hol~ (AsJltxs) reladve to the substrate in the substratebinding region. Interestingly, a number of negatively charged side chains were predicted to be present in or near the substrate-binding region of furin. These negatively charged residues in turin, which appeared to be absent in equivalent positions in the sabtilisins and thennitase, could interact directly witi,~ basic amino acid residues near the substrate cl:~avage site since they were predicted t9 be located in or near the S l, $2 and $4 binding pot:gets for lysine and/or arginine [441. It was hypothesized that such a high density of negative charges could contribute to a selectivity for positively charged substrate segments. It should be noted that most of these negatively charged residues appeared to be conserved among the various members of this novel family of endoproteases. This is in agreement with the general cleavage selectivity of these endoproteases for positively charged substrate segments.

Involvement in proprotein-processing of negatively charged amino acid residues in the substratebinding region of furin Furin-mediated proprotein cleavage within the constitutive secretory pathway was deduced to occur, predominantly at the consensus sequence Arg-X-Lys/ArgArg (RXK/RR) [36, 451. Analysis of furin-mediated

cleavage of substrate mutantsLf pro-vWF and profactor IX with non-conservat~e substitw, ions of the Arg residue at position P1 ~lso called -1 po',.;ition)'~ revealed that, in transient e~pression systems, exogenous furin could no longer cleave the subst, ate [10, 27, 29, 37]. Pro-vWF mutants involving non--onservarive substitutions at the P2 or P4 positions ;also called -2 and -4 positiens) were still processed t;y exogenous furin, although less efficient than wild-type pro-vWF [37, 42]. A P2 and P4 pro-v~/F dou]rqe mutant was not cleaved by furin [42]. A purified soluble forum of recombinant furin could not cleaw-, the precursor for anthrax toxin protective antigen ,n vitro in cage the Arg resi¢lue at PI or P4 was changed, whereas the Lys residue at the P2 position was apparently not essential [3(;, a 11. In conclusion, furin is also capable of cleavage, at RXXR and KR sequences, although less efficient than at RXK/RR sequences. It should be noted that similar results were recently obtained for exogenous PACE4, another endoprotease believed to be involved in processing within the constitutive secretory pathway [46, 47]. The results indicate that sequence requirements for cleavage by fuLin, at least in expression systems with high expression levels of exogenous furin or in vitro, are less stringent with respect to the presence of basic residues at the positions P2 and i~4, thaa with respect to an Arg residue at the P I position. Analysis of cleavage of pro-renin mutants in Chinese hamster ovary cells indicated that the sequence requirements for cleavage in the constitutive secretory pathway, presumably involving endogenous furin and/or PACE4, are also less restrictive than the initial consensus sequence RXK/RR suggested [48, 49]. In summary, the rules for suitable cleavage sequences proposed by Nakayama and colleagues are: I) the Arg residue at the PI position is essential; 2) in addition to the Arg residue at the PI position, at least two out of three basic residues at the P2, P4 and P6 positions are required for efficient cleavage (the presence of all the three basic residues results in the most efficient cleavage); and 3) at position PI' (also called + I position), a hydrophobic aliphatic amino acid residue is not suitable, The cleavage of P2 and P4 provWF mutants by exogenous furin or PACE4 is not conform the second rule proposed for endogenous cleavage in the constitutive pathway since no basic residue is present in pro-vWF at the P6 position. Possibly due to the high levels of exogenous furin or PACE4 compared to the endogenous expression levels, processing of the pro-vWF mutants can occur. However, it should be noted that native mouse prorenin2 contains only a KR sequence at the presumed cleavage site and is not cleaved by exogenous furin [45], conform the rules for endogenous cleavage. Similarly, anthrax toxin protective antigen with only basic amino acid residues at the P l, P2 and P3 pos-

213 itions was aiso not cleaved in vitro. Clearly, not only the presence or a~,,ence of certain basic residues in the substrate z,xe dczermining ~hether a substrate is cleaved or not. Ai'so secondary structures of the substrates near the ~cleavage site ~;hould be important. This is already implied by the fact that a hydrophobic aliphatic amino acid residt~e at the PI' position is not suitable. The analy~:~ tliscussed above clearly demonstrate the importance of the presence of basic amino acid residues at the P 1, P2, P4 and f'6 positions of the sub, strate cleavage site, although not for all the positions the requirements are equally strict. Considering the three-dimensional model of furin with its many negatively charged residues near or in the binding pockets for these basic residues, it is not difficult to envisage that at ~east some of these negatively charged residues ,,./

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Fig 4. Analysis of endoproteolytic processing of wild-type pro-vWF (R-S-K-R763) and pro-vWF cleavage mutants vWFK-2A (R-S-A-R763), a~ld vWFR-4A (A-S-K-R76~), by wild-type and four substrate-binding region mutants of human furin. For these studies [37], enzymes and substrates were expressed in PKI5 cells. Cells were first i~ected with recombinant vaccinia virus VV:T7, which encodes T7 RNA polymerase. Thereafter, cells were lipofected with plasmid DNA of which the eDNA insert, cloned downstream of a T7 promoter, was expressed due to transcription by T7 polymerase. The lipofected substrate DNAs were pro-vWF DNA (first row, vWF), pro-vWF mutant vWFK-2A DNA (second row, vWFK-2A), or pro-vWF mutant vWFR-4A DNA (third row, vWFR-4A), as already described above. Together with the lipofection of the pro-vWF-derived DNAs, DNAs encoding wild-type or one of the four substrata-binding region mutants of human furin were iipofected, as indicated above the lanes. In the first lane (-) no furin DNA was co-lipofected. Biosynthesis and processing of vWF-related proproteins wel~ studied in labelling experiments using [3.~S]-methionine. In the fourth row (Furin), biosynthesis and autocatalytical processing of wild-type and the substrate-binding region mutants of human furin as analyzed by immunoprecipitation are shown. The relative positions of pro-vWF and mature vWF and pro-furin and mature turin are indicated.

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Fig 3. Schematic representation of the substrate-binding region of the enzyme furin adapted from [44]. The positions of negativel/charged residues, predicted to be in or near to the substrate-binding region of furin and discussed in the text are indicated around the binding pockets S l, S2, and S4. These residues correspond to I)47, D84, EI23, D126, E129, El50, DI51, D152, D199, and D248. They are marked with a dot, an open circle or an asterisk, it. order to classify them with respect to the different effects mutations of these residues display (for explanation see text). The positions of the residues of the catalytic triad, D46, H87, and $261, and the oxyanion binding site N188 are boxed. The cleavage site of a hexapeptide substrate (P4-P3-P2-PIPI'-P2'), carboxy-terminal of PI, is indicated by a zig-zag line.

are directly interacting with basic amino residues of the substrate. It is not unreasonable to assume that they play a direct role in docking and subsequent cleavage of the substrate. To investigate this hypothesis, a panel of furin mutants was constructed, in which particular negatively charged residues predicted to be near or in the substrate binding region were replaced by neutral residues using site-directed mutagenesis t-,re'71, J. The prpdictpd.._. __ positiOnS nf . . . .these . . . . mutated residues relative to the S 1, $2, and $4 binding pockets of furin, the catalytic triad and the oxyanion hole, are schematically indicated in figure 3, which is adapted from Siezen [44]. Ir~ these mutants, only a single negatively charged residue was replaced, except in a double mutant, in which both Asp151 and Asp is2 were

214 replaced. Processing activity of these furin mutants was tested on wild-type pro-vWF (cleavage site: R-SK-R763,1,) and thrce pro-vWF mutants in which the prosequence preceding the cleavage site had been altered. These pro-vWF mutants with mutations at the P l, P2 and P4 positions included vWFR-IG (R-S-K-G7635), vWFK-2A (R-S-A-R763~) and vWFR-4A (A-S-KR763,1,). Biosynthesis and autocatalytic processing of wi!dotyp¢ human furin and the various furin mutants were also studied. Wild-type furin cleaved wild-type pro-vWF (360 kDa) in mature vWF (260 kDa) to completion, whereas the endogenous cleavage of provWF in the assay system used was very low. A major portion of the pro-furin (100 kDa) was processed to mature furin (_c~3kDa). As already mentioned above, wild-type furin also cleaved the P2 and P4 pro-vWF mutants (fig 4, lanes marked - and wt). As expected in light of the above mentioned rules, pro-vWF mutant vWFR-IG, with a mutation at the Pi position is not cleaved at all; not by wild-type furin nor by any of the furin mutants. This confirmed that the presence of a basic residue in the P I position is very critical. In the S 1 pocket, the interaction between a basic residue and negatively charged residues seems essential for docking and cleavage. Since Aspt99 is predicted to be the prime candidate for interaction with a PI basic residue in the S 1 pocket [44], it could be expected that furin mutant D I99G (Asp replaced by Gly at position 199) was not capable of cleaving of wild-type pro-vWF (nor any of the pro-vWF mutants). Here, interaction needed between the basic residue at the PI position and negatively charged residues in the S 1 pocket of furin is presumably impaired by the lack of Asp 199. Besides mutant DI99G, also double mutant DI51S/DI52G was not capable of cleaving any of the substrates used. Residues AsptSt and/or Aspt52 are probably involved in interactions near or within the $4 pocket [441. In the furin mutants lacking substrate processing activity, autocatalytic processing was also impaired. In conclusion, residue Asp 199 in the S 1 pocket and one or both of the residues AsplSt and Aspt52 in or near the $4 pocket, marked in figure 3 with open circles, seemed essential for furin activity. Furin mutants D84G, E1231 and EI50N showed no difference in cleaving wild-type pro-vWF or any of the pro-vWF mutants as compared to wild-type furin~ Also no difference in autocatalytic processing was seen. Thus, the residues AspS4, Glu123 and Glu 150, marked in figure 3 with an asterisk, seemed non~oo,.,+;nl ........ ,~. for c..-_ l..~m activity. Asp84 and GiulS0 are probably too far away for direct interaction with basic residues in a binding pocket. Glu 123 is probably located in or near the less essential $3 binding pocket [44]. The two classes of furin mutants discussed above showed either lack of processing activity or normal activity. However, remarkable results were obtained

with furin mutants D47T, D 126N, E129V and D248L corresponding to residues marked in figure 3 with a dot. The results are shown in figure 4 (lanes marked E I29V, D47T, D126N and D248L). Compared to wi!d4ype fudn, mutant E129V showed impaired processing of wild-type pro-vWF, hardly any processing of pro-vWF mutant vWFK-2A, whereas processing of pro-vWF mutant vWFR-4A by this furin mutant was as efficient as its processing by wild-type furin. It should be noted that furin mutant E129V processed the pro-vWF mutant vWFR-4A even better than wildtype pro-vWE The residue Glu 129 is predicted to be at the bottom of the $4 binding pocket of furin [44] and could have a critical position for interaction with the P4 arginine of the substrate. Results obtained with furin mutant E129V suggested that, as a result of the removal of this negatively charged residue, a basic residue at the P4 position of the substrate is no longer favourable. Furin mutant D47T was not able to cleave wild-type pro-vWF and pro-vWF mutant vWFR-4A, whereas it cleaved to some extent pro-vWF mutant vWFK-2A. Residue Asp 47 is predicted to be near the $2 binding pocket of furin [44] and could have a critical position for interaction with the P2 lysine. Results obtained with furin mutant D47T suggeste0 that, as a result of removal of this negatively charged residue, the basic residue at the P2 position in wild-type provWF and pro-vWF mutant vWFR-4A substrates severely impaired processing activity. This effect was found to be partially restored using the pro-vWF mutant vWFK-2A as substrate. Furin mutant D I26N showed some cleavage of wild-type pro-vWE but clearly less than wild-type furin. The mutant substrates were not cleaved at all. Residue Asp 126 is predicted to be at the entrance of the $4 binding pocket [44] and could have a critical position for interaction with the P4 arginine of the substrate. Furin mutants D47T, D126N and E I29V showed reduced autocatalytic processing. This is not unexpected since all three mutants showed at least some reduction in cleavage capability of the cleavage site of wild-type pro-vWF. In case of mutant E I29V, the amount of mature mutant furin was sufficient for almost complete processing of pro-vWF mutant vWFR-4A. In case of mutants D47T and D126N, the amount of mature mutant furin was sufficient for at least some processing of pro-vWF mutant vWFK-2A or wild-type pro-vWF respectively. Finally, furin mutant D248L showed no processing of pro-vWF substrates, but autocatalytic processing seemed to be normal. This result is difficult to explain. Although residue Asp 24s was predicted to be located some distance away from the catalytic groove, it could interact with two arginine residues of the furin enzyme itself (Siezen, Creemers and Van de Ven, submitted). Maybe impaired substrate processing is due to destabilization of the

215 enzyme, which apparently does not effect autocatalytic processing. The analysis of mutations of particular negatively charged residues, predicted to be located in or near the substrate binding region of furin, suggests that these residues are, as postulated, involved in interactions with basic amino acid residues of the substrate. In light of the recently proposed rules for cleavage of proproteins within the constitutive pathway [48, 49], it would be of interest to extend these studies of furin mutants towards the $6 binding pocket, since this pocket was also found to be of importance. Re-evaluation of the three-dimensional model of the catalytic domain of furin, taking into account the possibility of interactions between substrate basic amino acid residues and negatively charged residues in the $6 pocket of furin (Siezen, personal communications), suggests that Asp151 could interact With both the basic residue in the $4 and $6 pocket because of some rotational freedom° Asp 152 and/or Asp 157 are other candidates for interactions within or near the $6 binding pocket. Of interest to note here is the fact that the $6 binding pocket is at least partially comprised of an insertion in furin relative to subtilisin. This pocket is therefore not present in the prokaryotic subtilisins.

constitutive pathway two out of three should be a basic amino acid residue. It was shown that exogenous PC 1/PC3 cleaves according to these rules [50], indicative for the importance for processing in the regulated secretory pathway of negatively charged residues not only in the S 1 and $2 binding pockets but also in the $4 and $6 binding pockets. Contributions of additional binding subsites, such as $3 and $5, might also determine the observed differences in cleavage specificity of the proprotein processing enzymes. Alternatively, environmental factors may influence specificity, eg differences in local pH, substrate concentration or specific ion concentration.

Concluding remarks

References

Negatively charged amino acid residues in or near the substrate binding region of furin were found not only to be critical for cleavage activity but also for specificity. Substitution of Asp 47 by a threonine blocked cleavage of a substrate with a lysine at the P2 position, whereas substitution of Glu 129 by a valine changed the cleavage specificity towards a preferable, simple Lys-Arg pair. This indicates that by protein engineering the activity and the specificity of furin and other members of this family of endoproteases can be manipulated. Yet, we are still far from a complete understanding of the differences in substrate specificity of the different endoproteases. It is remarkable that negatively charged amino acid residues in position 126, 129, and 151 of human furin are conserved within the mammalian family of subtilisin-like endoproteases, even in those whose cleavage recognition site (RR and KR) does not seem to require an arginine at the P4 position (eg PC1/PC3 and PC2). However, recently proposed general rules concerning cleavage specificity for the regulated secretoi~y pathway are almost identical to the rules proposed for the constitutive secretory pathway [50]. The major difference is that for the regulated secretory pathway, only one of the three residues at the P2, P4 and P6 positions should be a basic amino acid residue, whereas in the

Acknowledgments This work was supported in part by the "Geconcerteerde Onderzoekacties 1992-1996', by Ec-contract BIOT-CT910302, and by the 'Stichting Technische Wetenschappen' (STW22.2726). This text presents results of the Belgian Programme on Interuniversity Poles of Attraction initiated by tt'.~ Belgian State, Prime Minister's Office, Science Policy Programming. The scientific responsibility is assumed by its authors. TAY Ayoubi is supported by a fellowship from the 'Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie (IWT)'.

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