Human Complement Factor I Does Not Require Cofactors for Cleavage of Synthetic Substrates

This information is current as Stefanos A. Tsiftsoglou and Robert B. Sim of October 2, 2021. J Immunol 2004; 173:367-375; ; doi: 10.4049/jimmunol.173.1.367 http://www.jimmunol.org/content/173/1/367 Downloaded from References This article cites 41 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/173/1/367.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Complement Factor I Does Not Require Cofactors for Cleavage of Synthetic Substrates1

Stefanos A. Tsiftsoglou2 and Robert B. Sim

Complement factor I (fI) plays a major role in the regulation of the . It circulates in an active form and has very restricted specificity, cleaving only or C4b in the presence of a such as (fH), type 1, membrane cofactor , or C4-binding protein. Using -7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of , but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino- Downloaded from

4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH3). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzene- sulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, ,only Cr2؉ and Fe3؉ were found to inhibit both the proteolytic and amidolytic activity of fI. The Journal of Immunology, 2004 173: 367Ð375. http://www.jimmunol.org/ he complement system is a major recognition and effector human fI (accession numbers: cDNA, Y00318; genomic, X78594) mechanism of innate immunity. Seven key serine pro- is localized on 4q25. T teases, (fD),3 MBL-associated serine fI has very restricted specificity limited to cleavage of arginyl (MASP)-2, C1s, C1r, factor B (fB), C2, and factor I (fI), play bonds in its natural protein substrates C3b and C4b. Cofactor pro- crucial roles in the generation of complement activities in the teins such as factor H (fH), complement receptor type 1, mem- phases of amplification and regulation of the cascade reactions (1, brane cofactor protein, or C4-binding protein are required for 2). Two additional homologues of MASP-2, namely, MASP-1 and cleavage. During natural substrate cleavage, fI forms a ternary MASP-3, have been identified, but their roles in complement ac- complex with the substrate and the cofactor (5). Certain aspects of tivation have yet to be determined. They all carry domains homol- the fI-cofactor-substrate complex remain unclear, such as whether by guest on October 2, 2021 ogous to the family and, with the exception of fD, addi- binding of the cofactor to both fI and the substrate is required for tional protein modules that influence the orientation and substrate orientation or is necessary for inducing appropriate con- localization of protein substrates and mediate complex formation formations in either the substrate or (1). through protein-protein interactions. fI is synthesized as a single polypeptide chain, which is glyco- fI plays an essential role in the modulation of the complement sylated and processed before secretion. No circulating zymogen cascade by the regulation of the C3 convertase of the classical and form has been identified. The mature protein consists of a N-ter- alternative activation pathways (3, 4). It is also essential for con- minal H chain with 317 aa residues and a C-terminal L chain with version of C3b into iC3b, a major . The encoding 244 residues (6, 7) that are covalently linked via a disulfide bond between residues Cys309 and Cys435 (S. A. Tsiftsoglou and A. C. Willis, unpublished data). Each chain contains three occupied N- Medical Research Council Immunochemistry Unit, Department of Biochemistry, Uni- linked sites contributing 20Ð25% (w/w) of the ap- versity of Oxford, South Parks Road, Oxford, United Kingdom parent protein molecular mass (8, 9). Analysis of the primary Received for publication March 15, 2004. Accepted for publication April 21, 2004. structure of fI reveals a unique linear arrangement of domains; a The costs of publication of this article were defrayed in part by the payment of page N-terminal fI membrane attack complex domain, an scavenger re- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ceptor cysteine-rich domain, and two class A low density lipopro- 1 This work was supported by the Medical Research Council (U.K.) and by a fees tein receptor domains in the noncatalytic H chain (6, 7, 10). The scholarship from the Department of Biochemistry, University of Oxford (to S.A.T.). C-terminal consists entirely of a trypsin-like L chain containing the 2 Address correspondence and reprint requests to Dr. Stefanos A. Tsiftsoglou, residues that define the His-Asp-Ser . In addition, Medical Research Council Immunochemistry Unit, Department of Biochemistry, residues are present that define the specificity pocket Asp189 and University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. E-mail address: 214 215 216 [email protected] the extended substrate binding sites Ser , Trp , and Gly . 3 Abbreviations used in this paper: fD, factor D; MASP, MBL-associated serine Among complement proteases, the domain of protease; fB, factor B; fI, factor I; fH, factor H; BoroMPG, Z-D-Phe-Pro- human fI is most similar to fD (28% sequence identity), methoxypropylboroglycinepinanediol ester; DFP, diisopropylfluorophosphate; but among all human serine proteases, it exhibits the closest sim- suramin, 8,8Ј-{carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1- phenylene)carbonylimino]}bis-1,3,5-naphthalenetrisulfonic acid; buffer A, 10 ilarity to human tissue (41% sequence iden- mM potassium phosphate, 0.5 mM EDTA, 0.1% Tween 20, pH 6.2; buffer B, 25 tity), and human plasma (37% sequence identity). fD mM Bicine, 0.5 mM EDTA, pH 8.25; AMC, 7-amino-4-methylcoumarin; ⑀ACA, ⑀-aminocaproic acid; SBTI, soybean ; LBTI, lima bean trypsin and fI both cleave their natural substrates in the presence of co- inhibitor type IIL. factors, and their rates of inhibition by substituted isocoumarins

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 368 SUBSTRATES AND INHIBITORS OF HUMAN COMPLEMENT fI are very low (11). There is structural evidence to suggest reversible substrate-induced conformational change within fD that may be required for optimal alignment of the catalytic triad (12, 13). This could account for the very low esterolytic activity (14) of isolated fD. A C3b-induced realignment of the catalytic site of fI has been proposed by Ekdahl et al. (15), on the basis of reactivity of fI with the serine protease inhibitor diisopropylfluorophosphate (DFP). To facilitate measurement of fI enzyme activity, we examined its amidolytic activity against 15 fluorogenic substrates. Kam et al. (11) examined 50 peptide thiobenzyl esters but found that none was cleaved by fI. Before this report, there has been no data de- scribing synthetic substrates for fI. 7-Amino-4-methylcoumarin (AMC) derivatives provide greater sensitivity in substrate assays than their thiobenzyl ester counterparts and have been used in stud- ies involving highly selective and low activity serine proteases, such as blood factors IXa (16) and VIIa (17) and the complement proteases fD, C2, and fB (14). Here, we provide the first report of the cleavage of synthetic substrates by fI in the absence of cofactors. Native fI cleaves pep- Downloaded from tides with a thrombin-like specificity, targeting Arg at the P1 po- sition, but with a lower catalytic activity than that of thrombin. The FIGURE 1. SDS-PAGE analysis of the protein preparations used in this work. fI, fH, and C3(NH ) were analyzed by SDS-10% PAGE under re- presence of cofactor has no significant influence on the enzymatic 3 duced and nonreduced conditions with Coomassie blue staining. Under turnover of these synthetic substrates, indicating that the cofactor reduced conditions, the heavily glycosylated (20Ð25% w/w) fI appears as does not alter the primary conformation of fI. two bands of which the first corresponds to the ϳ50-kDa H chain (HC) and the ϳ38-kDa L chain (LC) which is the serine protease domain. Similarly, ␣ ␤ http://www.jimmunol.org/ C3(NH3) appears as two bands corresponding to the - and -chains of the Materials and Methods molecule. fH appears as a single band under both conditions, whereas fI The following reagents were purchased from Sigma-Aldrich (St. Louis, and C3(NH3) under nonreduced conditions appear as single bands. Some MO): ⑀-aminocaproic acid (⑀ACA); antipain; aprotinin; barium chloride; aggregated fH is visible in both conditions. benzamidine; bestatin; chymostatin; DTT; ferric chloride; HEPES; 1,3,4,6- tetrachloro-3␣,6␣-diphenylglycoluril (Iodo-Gen); leupeptin; mercuric chloride; nickel chloride; BSA; pepstatin A; 1,10-; PMSF; Radiolabeling of C3(NH3) polyethylene glycol (PEG 3350); soybean trypsin inhibitor (SBTI). ␮ ␮ 125 Ammonium hydrogen carbonate, chromic potassium sulfate, EDTA, so- A sample of 50 g of C3(NH3) was labeled with 0.5 Ci of Na I using dium phosphate, manganese chloride, Tris, and zinc chloride were obtained the Iodo-Gen method (23). Nonincorporated iodide was removed by gel from BDH Laboratory Supplies (Poole, U.K.). Calcium chloride, glycine, filtration on a PD-10 column (Pharmacia) presaturated with 2 mg of BSA by guest on October 2, 2021 magnesium chloride, and sodium chloride were bought from Riedel-de Hae¬n and run in PBS (Dulbecco A; Oxoid, Basingstoke, U.K.), 0.5 mM EDTA, 125 (Seelze, Germany); cobalt chloride was purchased from Twinstar Chemicals pH 7.2. The specific radioactivity of the I-C3(NH3) was calculated as ϫ 6 ␮ (Harrow, U.K.). [4-(2-Aminoethyl)benzenesulfonyl fluoride ⅐ HCl (Pefabloc 2.3 10 dpm/min/ g. SC), N-␣-tosylglycyl-3-DL-amidinophenylalanine methyl ester (Pefabloc Xa), and N-␣-(2-naphthylsulfonylglycyl)-4-amidinophenylalaninepiperidide (Pefa- SDS-PAGE analysis bloc TH) were from Pentapharm (Basel, Switzerland)]; Z-D-Phe-Pro-me- The Laemmli system (24) was used for SDS-PAGE analysis, but the sam- thoxypropylboroglycinepinanediol ester (BoroMPG) from Calbiochem (EMD ple preparation, sample buffer composition, and Coomassie blue staining Biosciences, San Diego, CA), lima bean trypsin inhibitor type IIL (LBTI) from are as described by Fairbanks et al. (25). The composition of sample buffer Fluka (Buchs, Switzerland); suramin from Bayer (Leverkusen, Germany); was 0.2 M Tris, 8 M urea, 2% SDS, 0.002 M EDTA, pH 8.0, with 0.001% acrylamide and SDS from National Diagnostics (Atlanta, GA). Spectro/Por 6 bromphenol blue. dialysis tubing was from Medical Industries (Los Angeles, CA). Microfluor white plates were from Thermo Labsystems (Franklin, MA). Na125I was from 125 Cleavage of I-C3(NH3) by fI: a proteolytic assay for fI Amersham (Aylesbury, U.K.); human plasma was bought from HD Supplies (Aylesbury, U.K.). All chromatographic materials were purchased from Phar- The procedure is based on details given by Sim and Sim (26). Using as macia (Uppsala, Sweden). reaction buffer 10 mM potassium phosphate, 0.5 mM EDTA, 0.1% Tween AMC substrates were purchased from Calbiochem, ICN Biochemicals, 20, pH 6.2 (Buffer A) made 50 ␮g/ml with SBTI in 100 ␮l of reaction ϳ 125 American Diagnostica (Greenwich, CT), or Bachem (Budendorf, volume, 17,500 dpm of I-C3(NH3) (7.5 ng) was mixed with 62 ng of Switzerland). fH and variable amounts of fI starting from 40 ng. For controls 125I- 125 125 125 C3(NH3) only, I-C3(NH3) with fH, I-C3(NH3) with fI and I- C3(NH3) with fH and fI were prepared. All mixtures were incubated for 1 h Purification of at 37¡C, and the reactions were stopped by the addition of 50 ␮l of sample buffer made 40 mM with DTT. The mixtures were analyzed by SDS-8.5% Human C3 was purified using the method previously described by Dodds PAGE. Each gel was dried, and the results were obtained by autoradiog- 125 (18). The content of live C3, in which the thiol ester is intact, was judged raphy. An example can be found in Refs. 20 or 26. I-C3(NH3) is seen as by the absence of cleavage of the ␣-chain by fI in the presence of factor H a two-band pattern, the highly labeled 116-kDa ␣-chain and the 68-kDa ␤ ␣ (fH). Live C3 was converted to C3(NH3), an analog of C3b, after treatment -chain. On incubation with fI and fH, the -chain is cleaved into two with ammonium hydrogen carbonate, according to the report of Von Zab- fragments, one running with the ␤-chain and the other ϳ43 kDa. The rate ern et al. (19). Briefly, C3 (0.2Ð0.8 mg/ml concentration) was incubated of cleavage of the ␣-chain is proportional to the proteolytic activity of fI. with a final concentration of 0.2 M ammonium hydrogen carbonate for 90 Various compounds were tested as inhibitors in the proteolytic assay. Ϯ min at 37¡C, pH 8.0 0.1. C3(NH3) was then dialyzed against 20 mM Each compound was prepared from a stock solution using buffer A for all HEPES, 140 mM NaCl, 0.5 mM EDTA, pH 7.4; frozen in aliquots at 500 dilutions. fI (9.7 ng) was preincubated with the test compound at 37¡C for ␮g/ml; and stored at Ϫ80¡C. Human fI and fH were prepared according to 1 h before the addition of fH (31 ng). Incubation was continued for1hat 125 the method of Sim et al. (20). Both proteins were purified to homogeneity 37¡C, then I-C3(NH3) (17,500 dpm) was added, and the incubation at with some modifications ensuring the removal of trace contaminants. The pu- 37¡C was continued for 40 min. The final reaction volume was 100 ␮l. rified fI, fH, and C3 are shown in Fig. 1. Human C1 inhibitor was prepared Samples were analyzed by SDS-PAGE as described above, to measure the 125 according to the methods of Sim and Reboul (21) and Pilatte et al. (22). extent of cleavage. The positive control reaction of I-C3(NH3) with fH The Journal of Immunology 369

Table I. Summary of fluorogenic substrates examined for cleavage by human fI

Activity (fmol AMC released/ Substrates2a Substrate for min/␮g enzyme)b

Boc-Asp(OBzl)-Pro-Arg-AMCc,d Thrombin and trypsin 500 N-␣-test-Boc-Val-Pro-Arg-AMCe Thrombin 350 Z-Gly-Pro-Arg-AMCd Thrombin 250 Z-Leu-Leu-Arg-AMCd - 165 f Methylsulfonyl-D-Phe-Gly-Arg-AMC Tissue plasminogen activator 120 N-␣-test-Boc-Val-Leu-Lys-AMCe 30 Pro-Phe-Arg-AMCg Pancreatic and plasma 30 Boc-Phe-Ser-Arg-AMCd Factor XIa 30 Boc-Ile-Glu-Gly-Arg-AMCd Factors Xa, IX 20 Z-Gly-Gly-Arg-AMCd , tissue plasminogen activator, trypsin 20 and thrombin Z-Phe-Arg-AMCg Plasma and glandular kallikreins 10 Bz-Arg-AMCd Trypsin and papain 10 N-␣-test-Boc-Leu-Gly-Arg-AMCe Horseshoe crab clotting enzyme 0 MeOSuc-Ala-Ala-Pro-Val-AMCg 0 H-Gly-Arg-AMCd C 0

a All substrates were used at 25 ␮M final concentration and fI at 0.2 ␮M. b The values presented correspond to the mean of two determinations. Reproducibility was Ϯ5Ð10%. Downloaded from c Boc, test-Butyloxycarbonyl; OBzl, benzyl; MeOSuc, methoxysuccinyl, Z, carboxybenzyloxy, Ac, acetyl, Bz, benzoyl. d Bachem. e ICN Biochemicals. f American Diagnostica. g Calbiochem. http://www.jimmunol.org/ and fI did not reach complete cleavage during 40 min; thus, the data set Effects of pH, salt strength, and divalent ions on fI amidolytic could be used for comparison studies. Data were collected by the dissection activity of the dried autoradiograph and the measurement of the radioactivity of the cleavage products. The radioactivity in the 43-kDa (c) product was ex- The optimal pH for the AMC substrate cleavage was determined by the pressed as a percentage of the total radioactivity in each gel track. Aver- level of activity of fI on FGR-AMC substrate in a multiple buffer system. aged background from the negative controls was subtracted, and the data fI in 50 ␮l of 1 mM Tris-HCl, 25 mM NaCl, pH 7.4 was added to 100 ␮l 125 of 20 mM acetic acid, 20 mM MES, 20 mM HEPES, 20 mM Bicine, 100 obtained were expressed as percent inhibition using the I-C3(NH3) with fH and fI control as standard (0% inhibition). mM NaCl, pH 6.0Ð10.0; and 50 ␮l of FGR-AMC in water were added to give a final substrate concentration of 25 ␮M with 1 ␮M (88 ␮g/ml) fI in Synthetic substrates for fI a final reaction volume of 200 ␮l (Fig. 2).

To examine the effect of the salt strength on fI, FGR-AMC (final con- by guest on October 2, 2021 The following substrates were tested for cleavage by fI (Table I): R- centration, 25 ␮M) was added to fI (0.2 ␮M final concentration, 17.6 AMC; GR-AMC; GGR-AMC; LGR-AMC; FGR-AMC; IEGR-AMC; ␮g/ml) in buffer B (25 mM Bicine, 0.5 mM EDTA, pH 8.25) with NaCl FR-AMC; PFR-AMC; FSR-AMC; GPR-AMC; VPR-AMC; DPR- concentrations ranging from 0 to 1000 mM (Fig. 3). AMC; VLK-AMC; AAPV-AMC; and LLR-AMC. Each substrate, 25 The effects of Zn2ϩ,Mg2ϩ,Co2ϩ,Ca2ϩ,Mn2ϩ,Fe3ϩ,Ni2ϩ,Hg2ϩ, ␮M(final concentration) in 50 ␮l of 25 mM Bicine, 0.5 mM EDTA, pH ϩ ϩ ϩ Cu2 ,Ba2 and Cr2 ions on fI activity were also explored (Table III). 8.25 (buffer B) with 146 mM NaCl (final concentration), was added to 3.52 Each metal ion was tested individually at a final concentration of 1 mM on ␮g of fI in the same buffer in a Microfluor white plate. The final reaction fI (final concentration of 0.2 ␮M (17.6 ␮g/ml)) in 25 mM Bicine, pH 8.25. volume was 200 ␮l. The amidolytic activity of fI was measured using a microtiter plate reader (Fluoroskan; Thermo Life Sciences, Basingstoke, The fI and metal salts were incubated for1hat37¡C before the addition ␮ U.K.) by excitation at 355 nm and continuous monitoring of emission at of the DPR-AMC substrate (25 M final concentration). 460nmfor1hormore at 37¡C. Activity was expressed as the change in emission (⌬OD) per minute at a linear portion of the emission curve (initial rate). ⌬OD was converted to picomols of AMC released per minute per Heat stability of fI microgram of enzyme by use of a conversion factor calculated from com- plete substrate turnover. The heat stability of fI was tested in both proteolytic and amidolytic assays. Series of 2-fold serial dilutions of fI were assayed using FGR-AMC at Primarily a series of fI aliquots of 40 ng in 75 ␮l of buffer A were preheated 25 ␮M to determine a standard fI concentration that would yield a readily at temperatures of 37Ð91¡C for 30 min and tested for proteolytic activity at 125 measurable level of activity. The standard final concentration of fI used for 37¡C. The assay was stopped before complete cleavage of I-C3(NH3), most assays was 0.2 ␮M (17.6 ␮g/ml) unless otherwise stated. For selected and the autoradiograph produced was used for the measurement of the ⌲ ␣ 125 substrates, m and Vmax were obtained for both fI and human thrombin, degree of cleavage of the -chain of I-C3(NH3) for each sample using tested under identical conditions, using the Lineweaver-Burk plot method computer software (TotalLab; Nonlinear Dynamics, Newcastle-upon- (Table II). Tyne, U.K.).

Table II. Kinetic constants for the hydrolysis of selected substrates by human fI and thrombin

Vmax (pmol AMC ␮ ␮ Km ( M) released/min/ g enzyme)

Substrates fI Thrombin fI Thrombin

Boc-Asp(OBzl)-Pro-Arg-AMCa 14.6 19.9 0.79 49,751 N-␣-test-Boc-Val-Pro-Arg-AMC 27 9.6 0.69 32,051 Methylsulfonyl-D-Phe-Gly-Arg-AMC 128 25 0.87 3,940

a Boc, test-Butyloxycarbonyl; OBzl, benzyl. 370 SUBSTRATES AND INHIBITORS OF HUMAN COMPLEMENT fI

Table III. Summary of effects of divalent metal ions on amidolytic activity of fI

Metal Iona % Inhibitionb

No effect Zn2ϩ,Mg2ϩ,Co2ϩ,Ca2ϩ, 0Ð9 Mn2ϩ,Ba2ϩ,Ni2ϩ Inhibitory effect Cu2ϩ 23 Hg2ϩ 26 Cr2ϩ 54 Fe3ϩ 59

a All ions were used at 1 mM final concentration and factor I at 0.2 ␮M. The salt source for each ion is listed in Materials and Methods. b Values correspond to the mean of two determinations. Reproducibility was Ϯ5Ð10%.

FIGURE 2. Effect of pH on FGR-AMC cleavage by fI. FGR-AMC, 50 ␮l in water (25 ␮M final concentration), was added to 50 ␮loffIin1mM substrate (25 ␮M final concentration) in 50 ␮l of the same buffer. fH, ␮ ␮ Tris, 25 mM NaCl, 0.5 mM EDTA, (1 M; 88 g/ml final concentration) C3(NH3) and BSA were also tested in the absence of fI. Activity at 37¡C and 100 ␮l of 20 mM acetic acid, 20 mM MES, 20 mM HEPES, 20 mM was monitored as described above.

Bicine, 100 mM NaCl, pH 6.0Ð10.0. Cleavage in each sample was mon- Downloaded from itored and expressed as activity as described in Materials and Methods. Results fI activity on peptidyl-AMC substrates

FI aliquots of 3.52 ␮gin50␮l of buffer B were preheated at temper- The enzyme specificity of fI was explored by examining its cata- atures of 37Ð77¡C for 30 min before assay for the cleavage of the VPR- lytic capacity using the panel of 15 peptidyl-AMC substrates listed AMC substrate (25 ␮M final concentration). The final concentration of fI in Table I. A summary of the results obtained via spectrofluorim-

␮ ␮ http://www.jimmunol.org/ was 0.2 M (17.6 g/ml), and that of NaCl was 146 mM. etry is shown in Fig. 4A. The greatest release of the AMC fluoro- For each set of tests, the level of activity in each sample was expressed as percent total activity lost compared with the 37¡C control. phor was observed using the Asp-Pro-Arg derivative. Detectable enzyme activity was also observed using Val-Pro-Arg, Gly-Pro- Effect of inhibitors on fI activity Arg, Leu-Leu-Arg, and Phe-Gly-Arg. The Asp-Pro-Arg-AMC de- Inhibitors covering a wide spectrum of proteases were examined in the rivative is typically supplied as a substrate for use in determining amidolytic assay. Compounds at various concentrations were preincubated activity of thrombin and trypsin. The Val-Pro-Arg and Gly-Pro- for1hat37¡C with fI (final concentration, 0.2 ␮M (17.6 ␮g/ml)) in buffer Arg derivatives are routinely used to assay thrombin activity, and ␮ B before the assay of cleavage of FGR-AMC (25 M final concentration) the Phe-Gly-Arg derivative is used to detect the activity of tissue in the same buffer with 146 mM NaCl (final concentration). The concen- plasminogen activator. In the fI assays, cleavage was also seen tration of each compound used was chosen on the basis of solubility and by guest on October 2, 2021 mode of action of the compound. Compounds that caused significant in- with Val-Leu-Lys, Pro-Phe-Arg, Phe-Arg, Arg, Gly-Gly-Arg, Phe- hibition were explored further in separate dose dependence measurements Ser-Arg, and Ile-Glu-Gly-Arg substrates, although at lower levels. under conditions identical with those used for the initial screening. There was no detectable cleavage of Leu-Gly-Arg, Ala-Ala-Pro- Val, and Gly-Arg derivatives. All substrates tested, with the ex- Effects of C3(NH3) and fH on fI amidolytic activity ception of Val-Leu-Lys-AMC and Ala-Ala-Pro-Val-AMC, had ar- At a final concentration of 0.1 ␮M (8.8 ␮g/ml), fI was incubated alone or ginine at their P1 position. Val-Leu-Lys-AMC was cleaved at a in various combinations with up to 5-fold molar excess of fH, C3(NH3)or BSA (a negative control) in 150 ␮l of 25 mM Bicine, 0.5 mM EDTA, 152 comparatively low rate, and Ala-Ala-Pro-Val was not cleaved at mM NaCl, pH 8.25 for1hat37¡C, before the addition of the DPR-AMC all. Within this series of fI assays, there is a clear selectivity for

arginine at P1 position and a strong preference for proline at the P2 position. Only five substrates were cleaved at a significant rate, and

these had at the P3 position benzoylated Asp, Val, Gly, Leu, and Phe in order of decreasing rate (Table I). The characterized cleav- age sites in the natural substrates of fI are Pro-Ser-Arg, Leu-Leu- Arg in C3b and Thr-Gly-Arg, Arg-Gly-Arg in C4b. Of these nat- ural sequences, only Leu-Leu-Arg was tested, and its AMC derivative was cleaved by fI. A third site in iC3b, Leu-Gly-Arg, is controversially stated to be cleaved by fI. However, Leu-Gly-Arg AMC was not cleaved by fI. ⌲ The m and Vmax values for fI were calculated for three of selected synthetic substrates: Asp-Pro-Arg, Val-Pro-Arg, Phe-Gly- Arg (Fig. 4B), and are compared with the values obtained for na- ⌲ tive human thrombin (Table II). The m values calculated for fI and thrombin with Asp-Pro-Arg and Val-Pro-Arg derivatives are ⌲ very similar. The m of thrombin for the Phe-Gly-Arg-AMC sub- strate is 5-fold smaller than for fI. FIGURE 3. Effect of salt strength on FGR-AMC cleavage by fI. FGR- AMC, 50 ␮l in water (25 ␮M final concentration), was added to 50 ␮lof The Vmax values calculated for thrombin are, however, much fI in 1 mM Tris, 0.5 mM EDTA (0.2 ␮M; 17.6 ␮g/ml final concentration) higher in relation to these calculated for fI. For the Asp-Pro-Arg and 100 ␮l of 50 mM Bicine, 1.0 mM EDTA, pH 8.25, with a final NaCl and the Val-Pro-Arg substrates, the Vmax values of thrombin are 4 4 concentration of 0Ð1000 mM. Cleavage in each sample was monitored and 6.3 ϫ 10 - and 4.6 ϫ 10 -fold higher respectively than for fI. For ϫ expressed as activity as described in Materials and Methods. the Phe-Gly-Arg-AMC substrate, the Vmax for thrombin is 4.5 The Journal of Immunology 371

FIGURE 5. Investigation of possible effects of fH and C3(NH3)onthe amidolytic activity of fI. fI at a final concentration of 0.1 ␮M (8.8 ␮g/ml) was incubated alone (0 molar excess-positive control) or in various com- ϫ binations with fH, C3(NH3) or BSA (control) of up to 5 molar excess over fI, in 150 ␮l of 25 mM Bicine, 0.5 mM EDTA, 152 mM NaCl, pH Downloaded from 8.25, for1hat37¡C, before the addition of the DPR-AMC substrate (25 ␮ ␮ M final concentration) in 50 l of the same buffer. fH, C3(NH3), and BSA were also tested in the absence of fI (fH only, C3(NH3) only, and BSA only). Activity was monitored as described earlier. Background readings (buffer only control) were subtracted. http://www.jimmunol.org/ influence the amidolytic activity of fI in relation to small peptide- AMC substrates. This suggests that one of the roles of fH in the fI-mediated cleavage of C3b is to determine the substrate orienta- tion. The amidolytic activity assay is suitable for the detection and quantification of fI activity independent of cofactor proteins. FIGURE 4. A, Cleavage of peptidyl-AMC substrates by human fI. Fif- Effects of pH, salt strength, and divalent ions on factor I activity teen peptidyl AMC substrates (25 ␮M final concentration) in 50 ␮lof25 mM Bicine, 146 mM NaCl, 0.5 mM EDTA, pH 8.25, were added to 3.52 To determine the optimal conditions in which fI exhibits the high- by guest on October 2, 2021 ␮goffIin150␮l of the same buffer in Microfluor white plate wells and est level of amidolytic activity, a series of assays were conducted incubated for1hat37¡C. Cleavage was monitored continuously by mea- over a range of pH values, and also NaCl concentrations. In the suring emission at 460 nm, and the level of activity is shown as picomols range of pH values examined, pH 6Ð10, the activity appeared to of AMC released per minute per microgram of enzyme. Human thrombin rise steadily from pH 6.0 reaching a peak at 8.25, after which there (0.01 ␮g) was used as a calibration standard because it releases all AMC was a gradual decrease (Fig. 2). The bell-shaped curve centered present within 1 h. Substrates are as defined in Table I. B, Calculation of ⌲ around pH 8.0 is characteristic of many serine proteases. m and Vmax for selected substrates cleaved by fI. The values were calcu- lated using the Lineweaver-Burk plot method. Each calculation was based The activity of fI showed a moderate increase with the increase on a mathematical equation derived for the best fit trend line for each set of NaCl concentration between 0 and 145 mM reaching a maxi- of data corresponding to a particular substrate. The concentration of the mum in the region of physiological salt strength. At higher salt substrate ([S]) is micromolar, and the reaction velocity (v) is measured in concentrations, the activity showed a small decrease, reaching a picomols of AMC released per minute per microgram of enzyme. plateau from 200 to 600 mM and then a further small gradual decrease up to 1 M. The effect of salt is much more limited than that of pH. There is a Ͻ2-fold difference in amidolytic activity 103-fold higher than for fI. Thus, relative to thrombin, fI has sim- between optimum and very low or very high salt strength, whereas ilar affinity for these substrates, but much lower catalytic an ϳ6-fold difference in activity exists between those measured at efficiency. pH 6.0 and pH 8.25. In addition, the effect of divalent metal ions was also tested Investigation of effects of fH and C3(NH3) on the amidolytic (Table III). Of 11 metal ions examined, consisting of 10 bivalent activity of fI. and 1 trivalent species, Zn2ϩ,Mg2ϩ,Co2ϩ,Ca2ϩ,Mn2ϩ,Ba2ϩ, 2ϩ To explore whether fH or C3(NH3) affect the amidolytic activity of and Ni had no significant effect on the activity of fI. In contrast, fI, it was decided to test the levels of the amidolytic activity of fI Cu2ϩ,Hg2ϩ,Cr2ϩ, and Fe3ϩ had an inhibitory effect, with Cr2ϩ against the Asp-Pro-Arg-AMC substrate in the presence of either and Fe3ϩ causing almost 60% loss of activity. The others led to a

or both fH and C3(NH3). As shown in Fig. 5, excess fH, C3(NH3), decrease in activity ranging from 0 to 30% loss. From the selection or a mixture of both have no effect on the amidolytic activity of fI. of metal ions tested, Zn2ϩ,Cr2ϩ, and Fe3ϩ were also tested in the 2ϩ In addition, BSA used as a negative control also has no effect, and proteolytic assay using C3(NH3) as substrate (Table IV). Zn , these proteins have no intrinsic (or contaminating) amidolytic ac- which did not inhibit the activity of fI in the amidolytic assay, did 125 tivity. For cleavage of the natural substrates C3b and C4b by fI, it inhibit the breakdown of I-C3(NH3) at concentrations of 20 or has been hypothesized that protein-protein interactions among fI, 100 ␮M as previously reported by Crossley and Porter (27). This fH, and substrate are required to induce formation of fI into a is consistent with Zn2ϩ binding to fH, as reported by Sim et al. 2ϩ 3ϩ functionally active state. However, fH and C3(NH3) clearly do not (28), but not binding to fI. However, Cr and Fe inhibit both 372 SUBSTRATES AND INHIBITORS OF HUMAN COMPLEMENT fI

Table IV. Summary of effects of selected inhibitors on the proteolytic activity of fIa

Full Name Targets Final Concentration % Inhibition

Leupeptin Serine and cysteine proteases 10 ␮M29 Suramin Serine proteases 1 mM 90 Benzamidine Serine proteases 20 mM 0 Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester Thrombin 50 ␮M0 Antipain Serine and cysteine proteases 0.1 mM 0 PMSF Serine and cysteine proteases 1 mM 5 Aprotinin Serine proteases 0.5 ␮M23 Metal ion Salt source 2ϩ ␮ Zn ZnCl2 20 M21 2ϩ ␮ Zn ZnCl2 100 M59 3ϩ ⅐ ␮ Fe FeCl3 H2O 20 M25 3ϩ ⅐ ␮ Fe FeCl3 H2O 100 M23 2ϩ ⅐ ⅐ ␮ Cr Cr2(SO4)3 K2SO4 24H2O 20 M6 2ϩ ⅐ ⅐ ␮ Cr Cr2(SO4)3 K2SO4 24H2O 100 M43 a All dilutions from stock solutions were done with Buffer A.

the proteolytic and the amidolytic assays. The inhibitory effect of This is not the case here given that loss of activity in the amidolytic Downloaded from these two ions has not been tested before in the proteolytic assay. assay increases regularly with the increase in temperature. In the Crossley and Porter (27) had previously reported that Mg2ϩ, more detailed analysis of the proteolytic assay, the loss of activity 2ϩ 2ϩ 2ϩ 2ϩ Mn ,Ca ,Co , and Ni do not affect fI cleavage of C3(NH3) may occur in two stages, one between 37 and 48¡C and another in the presence of fH. from 61 to 73¡C. This is likely to be an indication of the behavior

of fI in complex formation with fH and C3(NH3), and indicative of Heat stability of fI a melting event occurring at two discrete points. http://www.jimmunol.org/ The heat stability of fI was tested in both the proteolytic and the amidolytic assays. Fig. 6 shows the loss of enzymatic activity over Effect of inhibitors on fI activity an extended range of temperatures of 37Ð91¡C. In both assays, fI To test the effect of inhibitors on fI, both the proteolytic and the Ͼ loses 50% of its activity between 37 and 65¡C, with 80% loss at amidolytic assays were used, the latter more extensively. In all 75¡C. In neither assay was the activity completely lost at temper- cases an excess of inhibitor was incubated with fI for1hat37¡C atures close to 80¡C, indicating that the serine protease domain of before the addition of the substrate. Inhibitors, both natural and fI shows a strong degree of thermal stability. In a multidomain synthetic, were first tested in the amidolytic assay (Table V). Se- protein, it might be expected that overall activity would be lost at lected compounds from this screen were subjected to further anal- by guest on October 2, 2021 the melting temperature of one particular domain, such that at one ysis with dose dependence responses in the amidolytic assay and in critical temperature there would be a very large loss of activity. the proteolytic assay for assessment of their impact on the physi- ological reaction (Table IV). From the compounds examined: EDTA-free protease inhibitor tablets (Boehringer-Mannheim, Mannheim, Germany; Pefabloc SC/Xa/TH; suramin; benzamidine; BoroMPG; and antipain, all strongly inhibited the amidolytic ac- tivity of fI (60Ð100% inhibition). In contrast, PMSF, aprotinin, leupeptin, SBTI, LBTI, hirudin, and ⑀ACA showed a more mod- erate inhibition of activity (10Ð40% inhibition). The remaining compounds, bestatin, C1 inhibitor, chymostatin, pepstatin A, and 1,10-phenanthroline, showed no evidence of inhibition. The dose- dependent nature of fI inhibition in the amidolytic assay by leu- peptin, antipain, BoroMPG, Pefabloc SC, suramin, and benzami- dine was demonstrated (Fig. 7). Most notably, antipain and leupeptin showed strong inhibition, 80% at 25 ␮M, although com- plete inhibition was not achieved. A 50% inhibition was observed with BoroMPG, Pefabloc SC, and Suramin at concentrations of 25, 40, and 125 ␮M, respectively. Benzamidine reached a plateau of FIGURE 6. Heat stability of fI. Two sets of fI samples, one in 10 mM inhibition close to 80% at 10 mM. From all the compounds ana- potassium phosphate, 0.5 mM EDTA, pH 6.2 (buffer A) and the other in 25 lyzed, only Pefabloc SC appeared to show complete enzyme mM Bicine, 0.5 mM EDTA, pH 8.25 (buffer B), 145 mM NaCl were inhibition. preheated at temperatures of 37Ð91¡C for 30 min. Each set was tested When leupeptin, antipain, BoroMPG, suramin, benzamidine, independently for its activity, the first in the proteolytic assay and the PMSF, and aprotinin were tested for inhibitory effects in the pro- second in the amidolytic assay. For the proteolytic assay, aliquots of 40 ng teolytic assay at the same concentrations as those used in the of fI in 75 ␮l of buffer A were exposed to temperatures of 37Ð91¡C and amidolytic assay (Table IV), only suramin, aprotinin, and leupep- tested for activity at 37¡C. The level of activity in each sample was ex- pressed as percent total activity lost compared with the 37¡C result set as tin gave results comparable with those observed in the amidolytic 0% loss. For the amidolytic activity assay, aliquots of 50 ␮l containing 0.8 assay. The amidolytic assay is done at pH 8.25, whereas the ␮M fI in buffer B were heated at 37Ð77¡C before testing on VPR-AMC. proteolytic assay is done at pH 6.2. Antipain, benzamidine, The final salt concentration was 145 mM NaCl. The activity data were BoroMPG, and PMSF worked less well than expected from the plotted as a function of temperature. results obtained in the amidolytic assay. Some of the results for the The Journal of Immunology 373

Table V. Summary of effects of inhibitors on the amidolytic assay of fI

Full Name Targets Concentration % Inhibitiona

Pefabloc-SC Serine proteases 0.25 mM 100 Pefabloc-Xa Factor Xa 0.25 mM 93 Suramin Serine proteases 1 mM 87 Benzamidine Serine proteases 20 mM 82 Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester Thrombin 50 ␮M82 Antipain Serine and cysteine proteases 0.1 mM 80 Pefabloc TH Thrombin 0.25 mM 58 PMSF Serine and cysteine proteases 1 mM 42 Aprotinin Serine proteases 0.5 ␮M37 Soybean trypsin inhibitor Serine proteases 50 ␮M37 Leupeptin Serine and cysteine proteases 10 ␮M24 Lima bean trypsin inhibitor IIL Serine proteases 50 ␮M20 Hirudin Thrombin 5 ATUb 19 ␧ACA Serine proteases 20 mM 10 Bestatin Metalloproteases 0.1 mM 0 C1 inhibitor Serine proteases 0.2 ␮M0 Chymostatin Serine and cysteine proteases 0.1 mM 0 Pepstatin A Aspartic acid proteases 1 ␮M0 1,10-Phenanthroline Metalloproteases 0.1 mM 0

a The values presented correspond to the mean of two determinations. Reproducibility was Ϯ5Ð10%. Boehringer-Mannheim Complete Mini EDTA-free protease inhibitor Downloaded from ϫ stock solution made by dissolving 1 tablet in 1.5 ml of H2O (according to manufacturer’s instructions) and diluting 4 into the final reaction volume inhibited the activity of fI by 100%. b One antithrombin U (ATU) neutralizes 1 NIH U of thrombin (fibrinogen assay) at 37¡C; 1 NIH U of thrombin clots a standard fibrinogen solution in 15 s at 37¡C; 5 ATU are expected to neutralize 2.5 ␮g of thrombin.

proteolytic assay are in agreement with observations made by Crossley and Porter (27), who reported no inhibition of the same http://www.jimmunol.org/ reaction by benzamidine or PMSF at concentrations of 5 and 0.1 mM, respectively. The same report noted that there are no effects from chelating agents such as EDTA or 1,10-phenanthroline at 50 mM and 1 mM, respectively. Discussion Although the physiological activity of fI has been broadly charac- terized, detailed descriptions of the potential range of its catalytic by guest on October 2, 2021 properties have not been available owing to the lack of identifiable synthetic substrates and high resolution structural data. Previously, information available on the substrate specificity and likely struc- ture of the active site has come from analysis of known cleavage sites in the natural substrates C3b or C4b and the work on the development of selective inhibitors (11, 15, 29, 30). The lack of reactivity of fI against thioester substrates reported previously (11) suggested the possibility that the catalytic L chain of fI is func- tionally inactive before substrate-induced conformation change. However, it is clear from the data presented here that fI has amidolytic activity in the absence of cofactors and natural sub- strates. This shows that the enzyme in its native state has a con- formation that accommodates substrate recognition and cleavage. The detection of amidolytic activity in the absence of fH and

C3(NH3) suggests that the requirement for fH in the cleavage of C3b in vivo is to support substrate orientation effects, but the pos- sibility that fI-cofactor interaction alters the conformation of fI to provide a secondary substrate has not been eliminated. This is in agreement with the data describing the interactions

within the ternary complex formed by fI, fH, and C3(NH3) (5, 31). In the experiment where all components, fI, fH, and C3(NH3), and

the synthetic substrate were present, C3(NH3) might be expected to act in competition with the synthetic substrate. However, the ␮ ratio of Asp-Pro-Arg (25 M) to C3(NH3) varied from 50- to FIGURE 7. Dose-dependent inhibition of fI by selected compounds as 250-fold molar excess, such that any competitive effect would be tested on the amidolytic assay. A, Leupeptin, antipain, BoroMPG, Pefabloc small. SC, suramin; B, Benzamidine. Different dilutions from stock preparations of each compound in 50 ␮l of 25 mM Bicine, 146 mM, 0.5 mM EDTA, pH 8.25, The substrate specificity patterns of fI are dependent on the ar- were coincubated for1hat37¡C with 100 ␮lof0.4␮M (35.2 ␮g/ml) fI in the chitecture of the serine protease domain of fI that aligns the desired same buffer. Each mixture was transferred to 50 ␮l of FGR-AMC substrate (25 arginyl bond to the active site area. The strong preference for Arg ␮ M final concentration) in the same buffer in a Microfluor white plate well. at P1 (Fig. 4A) is determined by the presence of an Asp residue at 374 SUBSTRATES AND INHIBITORS OF HUMAN COMPLEMENT fI position 189, located at the bottom of the specificity pocket that C1 inhibitor has the sequence Ser-Val-Ala-Arg, which is unlikely interacts electrostatically with positively charged residues like Arg to be compatible with the fI active site architecture. ⑀ACA, a lysine or Lys. For the P2 position, there is a preference for Pro. This analog, does cause slight inhibition, whereas benzamidine, an argi- 214 selectivity at S2 is under the influence of Ser . Surprisingly, at nyl analog, gave much stronger inhibition in accordance with the the natural cleavage sites, Gly, Ser, and Leu are preferred in P2 arginyl preference. (Pro-Ser-Arg, Leu-Leu-Arg in C3b and Thr-Gly-Arg, Arg-Gly- The moderate inhibition by SBTI and LBTI is probably due to Arg in C4b) instead of Pro. For position P3, little information can trace contaminants in the preparations or perhaps substrate com- be gained because, in the substrates that were cleaved best, the petition effects. Because these inhibitors form 1:1 complexes with residues at the P3 were diverse (Table I). In C3b and C4b cleavage their natural targets, it would be expected that if there was inhi- sites, Pro, Leu, Thr, and Arg all occur at the P3 position. It is bition, at such high molar excess (250ϫ for SBTI and LBTI), the difficult to judge which residues are preferred in P3, but those that percentage loss of activity should be 100%. This was not explored ␤ can form two antiparallel strand hydrogen bonds with the Gly at further. Hirudin gave a small effect at ϳ1:1 molar ratio. Of the position 216 (32) are likely to be the optimal. Pefabloc variants examined, Pefabloc TH is sold as a more selec- fI and thrombin appear to have similar synthetic substrate spec- tive inhibitor for thrombin, whereas Pefabloc Xa is relatively se- ificities, but they cleave at very different rates of catalysis (Table lective for factor Xa. Pefabloc SC was designed to cover a wider II). The similar substrate specificities can be attributed to homol- spectrum of proteases. Surprisingly, Pefabloc SC and Xa inhibited ogies within the substrate binding sites for both proteins (29, 30). fI much better than Pefabloc TH, despite indications that fI had Earlier studies on the specific activity of fI on C3(NH )inthe 3 specificity similar to that of thrombin. This variability could be due presence of fH had reported higher cleavage velocities than the ones reported here (33). The turnover rate reported for the cleavage to the presence of a Tyr at position 99 in Xa and fI, whereas Downloaded from thrombin has a Leu. The larger Tyr99 residue should make the aryl of the natural substrate C3(NH3)at37¡C in physiological condi- tions is 900 pmol of C3b cleaved per min per ␮g of fI, which is binding pocket of fI smaller than that of thrombin (30). Pefabloc significantly higher than the values described for the cleavage of SC inhibits fI very effectively, whereas the other wide range serine protease inhibitor, DFP, does not inhibit fI (27). tripeptides by fI. The Vmax values obtained for thrombin are still considerably higher. Although the two assays are significantly dif- Suramin inhibits well in both assays. It is a hexasulfonated ferent, making direct comparisons difficult, the efficiency of cleav- naphthylurea with a symmetrical structure (37) that has been used http://www.jimmunol.org/ age of the natural substrate is likely to be higher, because the for decades in the treatment of trypanosomiasis and onchocerciasis conformational effects that occur are likely to contribute to in- (38). It has proved useful as an antitumor agent (39) and as a potent creased efficiency. reversible inhibitor of the complement system (40) and of C3b Clotting proteases share ancestry with complement proteases breakdown (41). The first reports that suramin inhibits C3b break- (34, 35). The limited substrate range and low catalytic activity of down to iC3b were from Tamura and Nelson (42) and Lachmann fI is most likely related to its narrow activity against only two et al. (41). It was assumed then that binding of suramin to C3b natural substrates, in the presence of cofactors. A difference be- blocked the cleavage by fI, because treatment of C3b-coated cells tween fI and thrombin is the lack of of fI with suramin, before exposure to fI, inhibited breakdown of C3b to by guest on October 2, 2021 ϩ activity by Na ions. According to Dang and Di Cera (36), the iC3b (40). However, it is clear that suramin inhibits fI directly presence of a Tyr at position 225 in thrombin supports the binding (Table V). Suramin interacts with many proteins and proteases, ϩ ofaNa ion in a designated binding loop, whereas the replace- including trypsin (43). The similar degree of inhibition in both 225 ment of Tyr with a Pro in fI results in no such regulation. In assays indicates that suramin does not have strong direct effects on contrast, the proteolytic activity, which can be assessed only when fH or C3b. The affinity for suramin may be related to the basicity a cofactor protein and a protein substrate are present, is very de- of proteinases (39): the electrostatic interactions between the neg- pendent on salt strength. The rate of reaction decreases very sub- atively charged sulfonate groups of the polyanion and positively stantially between 10 and 150 mM (26). In addition, the pH opti- charged basic amino acids of proteases are likely to be the main Ͻ mum for this reaction when fH is the cofactor is low ( 5.5), but interactions in the enzyme-suramin complex. when CR1 is the cofactor, the optimum pH lies between 7 and 7.5 Leupeptin and antipain both inhibit fI effectively. Leupeptin has (26). These significant differences between the proteolytic and the sequence Leu-Leu-arginal which matches one of the natural amidolytic assays presumably reflect the weak ionic interactions cleavage sites in C3b and the synthetic substrate Leu-Leu-Arg- fI-cofactor, fI-substrate, and substrate-cofactor (5). The interac- AMC. Antipain has an Arg-Val-arginal sequence that resembles tions C3(NH )-fI, C3(NH )-fH, and fI-fH are stronger at low ionic 3 3 the Arg-Gly-Arg natural cleavage site in C4b. Leupeptin has pre- strengths and have pH optima lower than 6 (5). viously been described to inhibit thrombin as has aprotinin (44). The effects of inhibitors on the amidolytic assay confirmed the The moderate inhibition of fI by aprotinin that bears a Lys-Ala- serine protease nature of fI. When Crossley and Porter (27) tested a series of inhibitors on fI, they reported poor reactivity with sev- active site is consistent with the Arg preference of fI already dis- eral serine protease inhibitors. DFP, for example, did not inhibit. fI cussed. The BoroMPG inhibitor was tested as a representative of was characterized as a serine protease from sequence determina- the boropeptide thrombin inhibitors reported to cross-react with fI tion (6, 7). The activity of additional inhibitors reported in this (29, 30). It inhibits fI potently but is of limited solubility. paper is consistent with the nature of fI. As expected, bestatin, an Benzamidine and PMSF hardly inhibited in the proteolytic assay amino- and inhibitor, and 1,10-phenanthroline, a but were effective in the amidolytic assay. Crossley and Porter (27) metalloprotease inhibitor, did not inhibit at all, because no metals also found benzamidine ineffective in the proteolytic assay. Pos- 189 were found that regulate positively the activity of the enzyme. sibly its affinity with Asp is much lower compared with the Pepstatin, an aspartic acid protease inhibitor, and chymostatin, a natural substrate, and on complex formation the analog is likely specific inhibitor of , also did not inhibit. No inhibi- removed. The multiple interactions with substrate surfaces are tion was found with C1 inhibitor, a well-characterized serpin that likely to increase the stability and enhance the interactions that inhibits C1s, C1r, MASP-1, MASP-2, plasma kallikrein, factor result in the removal of the low molecular mass analog. Similar XIIa, and tissue plasminogen activator. The cleavage site in human effects were observed for the peptide analogues antipain and The Journal of Immunology 375

BoroMPG. Similarly to benzamidine, the fI-C3(NH3) complex for- 17. Kam, C. M., G. P. Vlasuk, D. E. Smith, K. E. Arcuri, and J. C. Powers. 1990. mation is likely to remove the peptide analogs due to higher af- Thioester chromogenic substrates for human factor VIIa: substituted isocouma- rins are inhibitors of factor VIIa and in vitro anticoagulants. Thromb. Haemost. finity for the natural substrate. In both tests, aprotinin was found to 64:133. inhibit fI to a similar, moderate extent. Differences between the 18. Dodds, A. W. 1993. Small-scale preparation of complement components C3 and two assays may also be partly due to a pH effect. More extensive C4. Methods Enzymol. 223:46. 19. von Zabern, I., E. L. Bloom, V. Chu, and I. Gigli. 1982. The fourth component tests of affinities at different pH and salt values are required for a of human complement treated with amines or chaotropes or frozen-thawed (C4b- fuller investigation of the effects of inhibitors. like C4): interaction with C4 binding protein and cleavage by C3b/C4b inacti- vator. J. Immunol. 128:1433. In conclusion, this work identifies means to assay fI alone with- 20. Sim, R. B., A. J. Day, B. E. Moffatt, and M. Fontaine. 1993. Complement factor out a cofactor. The novelty of the exploration of the active site of I and cofactors in control of complement system convertase . Methods fI with substrates and inhibitors provides valuable information to- Enzymol. 223:13. 21. Sim, R. B., and A. Reboul. 1981. Preparation and properties of human C1 in- ward the elucidation of the configuration of the active site and the hibitor. Methods Enzymol. 80(Pt. C):43. specificity regions. Mapping of certain specificity regions and 22. Pilatte, Y., C. H. Hammer, M. M. Frank, and L. F. Fries. 1989. A new simplified characterization of the interactions within and around the active procedure for C1 inhibitor purification. A novel use for jacalin-agarose. J. Im- munol. Methods 120:37. site can provide powerful information that may guide the effort for 23. Fraker, P. J., and J. C. Speck, Jr. 1978. Protein and cell membrane iodinations synthesis of specific inhibitor compounds that could have impor- with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3␣,6␣-diphenylgly- tant medical potential. Future analysis of a three-dimensional coluril. Biochem. Biophys. Res. Commun. 80:849. 24. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the structure will complement the current observations. head of bacteriophage T4. Nature 227:680. 25. Fairbanks, G., T. L. Steck, and D. F. Wallach. 1971. Electrophoretic analysis of Acknowledgments the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606. Downloaded from We thank Dr. D. A. Mitchell for his help in composition of the manuscript. 26. Sim, E., and R. B. Sim. 1983. Enzymic assay of C3b receptor on intact cells and solubilized cells. Biochem. J. 210:567. References 27. Crossley, L. G., and R. R. Porter. 1980. Purification of the human complement 1. Arlaud, G. J., J. E. Volanakis, N. M. Thielens, S. V. Narayana, V. Rossi, and control protein C3b inactivator. Biochem. J. 191:173. Y. Xu. 1998. The atypical serine proteases of the complement system. Adv. Im- 28. Sim, R. B., V. Malhotra, J. Ripoche, A. J. Day, K. J. Micklem, and E. Sim. 1986. munol. 69:249. Complement receptors and related complement control proteins. Biochem. Soc. 2. Sim, R. B., and S. A. Tsiftsoglou. 2004. Proteases of the complement system. Symp. 51:83.

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