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For Furin: Application As an Antipathogenic Agent Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7293–7298, June 1998 Biochemistry a1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent FRANC¸OIS JEAN†,KORI STELLA‡,LAUREL THOMAS†,GSEPING LIU†,YANG XIANG†,ANDREW J. REASON§, AND GARY THOMAS†¶ †Vollum Institute and ‡Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201; and §M-Scan Inc., West Chester, PA 19380 Communicated by Donald F. Steiner, University of Chicago, Chicago, IL, April 13, 1998 (received for review February 4, 1998) ABSTRACT The important role of furin in the proteolytic exotoxin A (PEA), and diphtheria and Shiga toxins] is required activation of many pathogenic molecules has made this endo- for their cytotoxicity (6). protease a target for the development of potent and selective The discovery that a diverse group of viral and bacterial antiproteolytic agents. Here, we demonstrate the utility of the pathogens requires furin for proteolytic activation has fostered protein-based inhibitor a1-antitrypsin Portland (a1-PDX) as an the development of furin inhibitors. These include active-site- antipathogenic agent that can be used prophylactically to block directed chloromethyl ketone inhibitors (7, 8), reversible peptide furin-dependent cell killing by Pseudomonas exotoxin A. Bio- inhibitors (9, 10), and several protein-based inhibitors (11–14). chemical analysis of the specificity of a bacterially expressed His- The protein-based inhibitors include a recently described native a a y and FLAG-tagged 1-PDX ( 1-PDX hf) revealed the selectivity serpin, PI8 (14), which contains two consensus furin sites, -Arg- a y 339 339 342 of the 1-PDX hf reactive site loop for furin (Ki, 600 pM) but not Asn-Ser-Arg - and -Arg -Cys-Ser-Arg -, in its reactive site for other proprotein convertase family members or other unre- domain. Other protein-based inhibitors represent engineered a y lated endoproteases. Kinetic studies show that 1-PDX hf in- variants of either the turkey ovomucoid third domain (-Ala-Cys- 18 3 18 a hibits furin by a slow tight-binding mechanism characteristic of Thr-Leu - -Arg-Cys-Lys-Arg -) (11), 2-macroglobulin serpin molecules and functions as a suicide substrate inhibitor. (-Gly-Phe-Tyr-Glu686-Ser-Asp- 3 -Arg-Ser-Lys-Arg686-Ser- a y a a a Once bound to furin’s active site, 1-PDX hf partitions with Leu-) (13), or 1-antitrypsin ( 1-AT). The latter variant, 1-AT equal probability to undergo proteolysis by furin at the C- Portland (a -PDX), is distinct in that it contains a single minimal 2 1 terminal side of the reactive center -Arg355-Ile-Pro-Arg358- or to furin consensus motif in its reactive site loop (RSL; Ala355-Ile- form a kinetically trapped SDS-stable complex with the enzyme. Pro-Met358- 3 -Arg355-Ile-Pro-Arg358-) (12). This partitioning between the complex-forming and proteolytic a -PDX is a potent inhibitor of furin (IC 5 0.6 nM) and when a y 1 50 pathways contributes to the ability of 1-PDX hf to differentially expressed in cells (either by stable or transient transfection), inhibit members of the proprotein convertase family. Finally, we blocks the processing of HIV-1 gp160 and measles virus-F and a o propose a structural model of the 1-PDX-reactive site loop that correspondingly inhibits virus spread (12, 15). However, relative explains the high degree of enzyme selectivity of this serpin and to the chymotrypsin superfamily of serine proteases, little is which can be used to generate small molecule furin inhibitors. known regarding the mechanism of inhibition of subtilase super- a family members, including furin, by 1-AT or its engineered a The recent discovery of a novel family of mammalian subtilisin- variants. In addition, although 1-PDX does not inhibit either related proprotein convertases (PCs) that reside in the secretory elastase or thrombin (12), the selectivity of this furin-directed pathway has led to a more detailed understanding of the molec- inhibitor for the other PCs has not been established. Finally, a ular basis of proprotein maturation (1). Members of the PC family whereas genome expression of 1-PDX effectively blocks pro- identified to date include furin, PACE-4, PC2, PC1y3 (hereafter protein maturation (12, 15), it remains to be determined whether termed PC3), PC4, PC5y6A and B (hereafter termed PC6A and the recombinant protein can be used as a therapeutic agent. y y a PC6B), and LPC PC7 PC8 (hereafter termed PC7) (2). Here, we report the mechanism of furin inhibition by 1-PDX y a One PC, furin, trafficks through the trans-Golgi network and the intrinsic selectivity of 1-PDX for furin but not for other a endosomal system where it cleaves a wide range of proproteins at PCs. Furthermore, we show that 1-PDX can be used prophy- 2 the consensus sequence -Arg-Xaa-LysyArg-Arg- (3). Muta- lactically to block cell killing by PEA, a clinically important tional analyses showed that the minimal consensus sequence pathogen gene product. Our model of the a -PDX-RSL provides 2 1 required for furin processing is -Arg-Xaa-Xaa-Arg- (4). By a basis for determining the interactions important for substrate contrast, PC2 and PC3 reside in the regulated secretory pathway binding and enzyme selectivity. How this model may facilitate of neuroendocrine cells where they process prohormones at development of small molecule therapeutics is discussed. -LysyArg-Arg- sites (1, 5). The roles of the remaining PCs have yet to be determined. In addition to endogenous proproteins, many pathogens re- MATERIALS AND METHODS quire furin to become active (3). For example, several viral coat Materials. pGlu-Arg-Thr-Lys-Arg-4-methylcoumaryl-7-amide proteins including HIV type 1 (HIV-1) gp160, Newcastle-disease (pERTKR-MCA) was obtained from Peptides International, virus Fo, measles virus Fo, and human cytomegalovirus glycop- N-tert-butoxycarbonyl-Asp(benzyl)-Pro-Arg-MCA and de- rotein B, require processing by furin in the biosynthetic pathway canoyl-Arg-Val-Lys-Arg-CH2Cl (Dec-RVKR-CH2Cl) from to generate infectious virions (3). Similarly, cleavage of many bacterial toxins by furin at the plasma membrane (e.g., anthrax a a a y y Abbreviations: 1-PDX, 1-antitrypsin Portland; 1-PDX hf, His- and toxin) and or in endosomal compartments [e.g., Pseudomonas FLAG-tagged a1-PDX; RSL, reactive site loop; PC, proprotein con- vertase; PEA, Pseudomonas exotoxin A; a1-AT, a1-antitrypsin; pER- The publication costs of this article were defrayed in part by page charge TKR-MCA, pGlu-Arg-Thr-Lys-Arg-4-methylcoumaryl-7-amide; Dec- RVKR-CH Cl, decanoyl-Arg-Val-Lys-Arg-CH Cl; a -PIT, a - payment. This article must therefore be hereby marked ‘‘advertisement’’ in 2 2 1 1 antitrypsin Pittsburgh; RP-HPLC, reverse-phase HPLC; SI, accordance with 18 U.S.C. §1734 solely to indicate this fact. stoichiometry of inhibition. © 1998 by The National Academy of Sciences 0027-8424y98y957293-6$2.00y0 ¶To whom reprint requests should be addressed. e-mail: thomasg@ PNAS is available online at http:yywww.pnas.org. ohsu.edu. 7293 Downloaded by guest on September 24, 2021 7294 Biochemistry: Jean et al. Proc. Natl. Acad. Sci. USA 95 (1998) Bachem, thrombin (EC 3.4.21.5) from Sigma, and mAb M2 from Cell Culture. BSC-40, AtT20, LoVo, and A7 cells were Kodak. grown as described (15–17). PEA assays were performed as His-yFLAG-Tagged a1-AT Variants. The DNA sequence en- described (17). a coding the human 1-AT signal peptide in pBS-AT (12) was Vaccinia Virus Recombinants. To monitor enzyme expression replaced with sequences encoding the epitope(FLAG)-tag (un- the FLAG tag was inserted by loop-in mutagenesis using muta- derlined) by insertion of the annealed complimentary oligos genic primers C terminal to the proregion cleavage site of each 59-CTAGAGGATCCCATGGACTACAAGGACGACGATG- PC (except for PC2) as follows: FLAG-tagged (underline) human ACAAGGAA-39 and 59-GATCTTCCTTGTCATCGTCGTC- PACE-4 (hPACE-4yf), 59-CGAAGGGTGAAGA-GAGACTA- CTTGTAGTCCATGGGATCCT-39. The resulting cDNA was CAAGGACGACGATGACAAGCAGGTGCGAAGTGAC- a y 9 149 y 9 subcloned into pDS56–6His to generate pDS56 1-AT hf. To -3 (C terminal to Arg ) (18); murine PC3 (mPC3 f), 5 -AG- a y generate pDS56 1-PDX hf, the DNA sequences encoding the AGAGAAGTAAACGTGACTACAAGGACGACGATGAC- 9 110 RSL were replaced by the complimentary oligos (59-GGGGC- AAGTCAGTTCAAAAAGACT-3 (C terminal to Arg ) (19); y 9 CATGTTTTTAGAGCGCATACCCA-39 and 59-GATCTGG- murine PC6B (mPC6B f), 5 -AAAAAGAACCAAGAGGGA- GTATGCGCTCTAAAAACATGGCCCCTGCA-39) coding CTACAAGGACGACGATGACAAGGATTATGACCTCA- a GCC-39 (C terminal to Arg116) (20). A secreted soluble FLAG- for Arg (bold) in positions P4 and P1. To generate pDS56 1- y PITyhf, complimentary oligos (59-GGGGCCATGTTTTTAG- tagged human PC7 (hPC7 f) was generated by inserting the 9 9 epitope tag C terminal to Arg141 (21) by using the mutagenic AGGCCATACCCA-3 and 5 -GATCTGGGTATGGCCTCTA- 9 AAAACATGGCCCCTGCA-39) encoding a P1 Arg were used. primer 5 -CTGCTAAGGCGGGCTAAGCGCGACTACAA- The resulting ORFs directed cytosolic expression of the recom- GGACGACGATGACAAGTCGGTCCACTTCAACGACC- -39 and a second primer 59-CCCAACACCCTCAAGTAGGCC- binant proteins initiating with a Met followed by the His and 9 FLAG tags and the mature sequences of either a -PDXyhf or TAGCTGGTAGGCTGTTTC-3 to insert a stop codon (bold) in 1 place of Thr667. Generation of vaccinia virus recombinants ex- a -PITyhf (see Fig. 1A). 1 pressing each of the PCs was performed as described previously Expression and Purification of a1-PDXyhf. a -AT variants 1 (22). The human fur713tyf (hereafter termed hfurinyf) and were expressed in Escherichia coli strain BL21 transformed with human PC2 (hPC2) constructs were described previously (5, 22). either pDS56a -PDXyhf or pDS56a -PITyhf. Protein expression 1 1 Expression of Recombinant PCs. Each PC was produced by was induced by addition of 1 mM isopropyl b-D-thiogalactoside, infecting cultured cells with the corresponding vaccinia virus and cultures were grown overnight at 31°C.
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