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Human Complement Factor I Does Not Require Cofactors for Cleavage of Synthetic Substrates ¹

Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, United Kingdom

	Abstract

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Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-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 thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases 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-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 benzenesulfonyl 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The complement system is a major recognition and effector mechanism of innate immunity. Seven key serine proteases, factor D (fD), ³ MBL-associated serine protease (MASP)-2, C1s, C1r, factor B (fB), C2, and factor I (fI), play crucial roles in the generation of complement activities in the phases of amplification and regulation of the cascade reactions (1 2). Two additional homologues of MASP-2, namely, MASP-1 and MASP-3, have been identified, but their roles in complement activation have yet to be determined. They all carry domains homologous to the trypsin family and, with the exception of fD, additional protein modules that influence the orientation and localization of protein substrates and mediate complex formation through protein-protein interactions.

fI plays an essential role in the modulation of the complement cascade by the regulation of the C3 convertase of the classical and alternative activation pathways (3, 4). It is also essential for conversion of C3b into iC3b, a major opsonin. The gene encoding human fI (accession numbers: cDNA, Y00318; genomic, X78594) is localized on chromosome 4q25.

fI has very restricted specificity limited to cleavage of arginyl bonds in its natural protein substrates C3b and C4b. Cofactor proteins such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein are required for cleavage. During natural substrate cleavage, fI forms a ternary complex with the substrate and the cofactor (5). Certain aspects of the fI-cofactor-substrate complex remain unclear, such as whether binding of the cofactor to both fI and the substrate is required for substrate orientation or is necessary for inducing appropriate conformations in either the substrate or enzyme (1).

fI is synthesized as a single polypeptide chain, which is glycosylated and processed before secretion. No circulating zymogen form has been identified. The mature protein consists of a N-terminal H chain with 317 aa residues and a C-terminal L chain with 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-linked glycosylation sites contributing 20–25% (w/w) of the apparent protein molecular mass (8, 9). Analysis of the primary structure of fI reveals a unique linear arrangement of domains; a N-terminal fI membrane attack complex domain, an scavenger receptor cysteine-rich domain, and two class A low density lipoprotein receptor domains in the noncatalytic H chain (6, 7, 10). The C-terminal consists entirely of a trypsin-like L chain containing the residues that define the His-Asp-Ser catalytic triad. In addition, residues are present that define the specificity pocket Asp¹⁸⁹ and the extended substrate binding sites Ser²¹⁴, Trp²¹⁵, and Gly²¹⁶.

Among complement proteases, the serine protease domain of human fI is most similar to fD (28% amino acid sequence identity), but among all human serine proteases, it exhibits the closest similarity to human tissue plasminogen activator (41% sequence identity), and human plasma kallikrein (37% sequence identity). fD and fI both cleave their natural substrates in the presence of cofactors, and their rates of inhibition by substituted isocoumarins 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 describing 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 studies involving highly selective and low activity serine proteases, such as blood coagulation 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 peptides with a thrombin-like specificity, targeting Arg at the P₁ position, but with a lower catalytic activity than that of thrombin. The presence of cofactor has no significant influence on the enzymatic turnover of these synthetic substrates, indicating that the cofactor does not alter the primary active site conformation of fI.

	Materials and Methods

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The following reagents were purchased from Sigma-Aldrich (St. Louis, MO): -aminocaproic acid (ACA); antipain; aprotinin; barium chloride; 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-phenanthroline; PMSF; polyethylene glycol (PEG 3350); soybean trypsin inhibitor (SBTI).

Ammonium hydrogen carbonate, chromic potassium sulfate, EDTA, sodium phosphate, manganese chloride, Tris, and zinc chloride were obtained from BDH Laboratory Supplies (Poole, U.K.). Calcium chloride, glycine, magnesium chloride, and sodium chloride were bought from Riedel-de Haën (Seelze, Germany); cobalt chloride was purchased from Twinstar Chemicals (Harrow, U.K.). [4-(2-Aminoethyl)benzenesulfonyl fluoride · HCl (Pefabloc SC), N--tosylglycyl-3-DL-amidinophenylalanine methyl ester (Pefabloc Xa), and N--(2-naphthylsulfonylglycyl)-4-amidinophenylalaninepiperidide (Pefabloc TH) were from Pentapharm (Basel, Switzerland)]; Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester (BoroMPG) from Calbiochem (EMD Biosciences, San Diego, CA), lima bean trypsin inhibitor type IIL (LBTI) from Fluka (Buchs, Switzerland); suramin from Bayer (Leverkusen, Germany); acrylamide and SDS from National Diagnostics (Atlanta, GA). Spectro/Por 6 dialysis tubing was from Medical Industries (Los Angeles, CA). Microfluor white plates were from Thermo Labsystems (Franklin, MA). Na¹²⁵I was from Amersham (Aylesbury, U.K.); human plasma was bought from HD Supplies (Aylesbury, U.K.). All chromatographic materials were purchased from Pharmacia (Uppsala, Sweden).

AMC substrates were purchased from Calbiochem, ICN Biochemicals, American Diagnostica (Greenwich, CT), or Bachem (Budendorf, Switzerland).

Purification of proteins

Human C3 was purified using the method previously described by Dodds (18). The content of live C3, in which the thiol ester is intact, was judged by the absence of cleavage of the -chain by fI in the presence of factor H (fH). Live C3 was converted to C3(NH₃), an analog of C3b, after treatment with ammonium hydrogen carbonate, according to the report of Von Zabern et al. (19). Briefly, C3 (0.2–0.8 mg/ml concentration) was incubated with a final concentration of 0.2 M ammonium hydrogen carbonate for 90 min at 37°C, pH 8.0 ± 0.1. C3(NH₃) was then dialyzed against 20 mM HEPES, 140 mM NaCl, 0.5 mM EDTA, pH 7.4; frozen in aliquots at 500 µg/ml; and stored at –80°C. Human fI and fH were prepared according to the method of Sim et al. (20). Both proteins were purified to homogeneity with some modifications ensuring the removal of trace contaminants. The purified fI, fH, and C3 are shown in Fig. 1. Human C1 inhibitor was prepared according to the methods of Sim and Reboul (21) and Pilatte et al. (22).

View larger version (84K): [in this window] [in a new window]   FIGURE 1. SDS-PAGE analysis of the protein preparations used in this work. fI, fH, and C3(NH3) were analyzed by SDS-10% PAGE under reduced and nonreduced conditions with Coomassie blue staining. Under reduced conditions, the heavily glycosylated (20–25% w/w) fI appears as two bands of which the first corresponds to the 50-kDa H chain (HC) and the 38-kDa L chain (LC) which is the serine protease domain. Similarly, C3(NH3) appears as two bands corresponding to the - and -chains of the molecule. fH appears as a single band under both conditions, whereas fI and C3(NH3) under nonreduced conditions appear as single bands. Some aggregated fH is visible in both conditions.

A sample of 50 µg of C3(NH₃) was labeled with 0.5 µCi of Na¹²⁵I using the Iodo-Gen method (23). Nonincorporated iodide was removed by gel filtration on a PD-10 column (Pharmacia) presaturated with 2 mg of BSA and run in PBS (Dulbecco A; Oxoid, Basingstoke, U.K.), 0.5 mM EDTA, pH 7.2. The specific radioactivity of the ¹²⁵I-C3(NH₃) was calculated as 2.3 x 10⁶ dpm/min/µg.

SDS-PAGE analysis

The Laemmli system (24) was used for SDS-PAGE analysis, but the sample preparation, sample buffer composition, and Coomassie blue staining are as described by Fairbanks et al. (25). The composition of sample buffer was 0.2 M Tris, 8 M urea, 2% SDS, 0.002 M EDTA, pH 8.0, with 0.001% bromphenol blue.

Cleavage of ¹²⁵I-C3(NH₃) by fI: a proteolytic assay for fI

The procedure is based on details given by Sim and Sim (26). Using as reaction buffer 10 mM potassium phosphate, 0.5 mM EDTA, 0.1% Tween 20, pH 6.2 (Buffer A) made 50 µg/ml with SBTI in 100 µl of reaction volume, 17,500 dpm of ¹²⁵I-C3(NH₃) (7.5 ng) was mixed with 62 ng of fH and variable amounts of fI starting from 40 ng. For controls ¹²⁵I-C3(NH₃) only, ¹²⁵I-C3(NH₃) with fH, ¹²⁵I-C3(NH₃) with fI and ¹²⁵I-C3(NH₃) with fH and fI were prepared. All mixtures were incubated for 1 h at 37°C, and the reactions were stopped by the addition of 50 µl of sample buffer made 40 mM with DTT. The mixtures were analyzed by SDS-8.5% PAGE. Each gel was dried, and the results were obtained by autoradiography. An example can be found in Refs. 20 or26 . ¹²⁵I-C3(NH₃) is seen as a two-band pattern, the highly labeled 116-kDa -chain and the 68-kDa -chain. On incubation with fI and fH, the -chain is cleaved into two fragments, one running with the -chain and the other 43 kDa. The rate of cleavage of the -chain is proportional to the proteolytic activity of fI.

Various compounds were tested as inhibitors in the proteolytic assay. Each compound was prepared from a stock solution using buffer A for all dilutions. fI (9.7 ng) was preincubated with the test compound at 37°C for 1 h before the addition of fH (31 ng). Incubation was continued for 1 h at 37°C, then ¹²⁵I-C3(NH₃) (17,500 dpm) was added, and the incubation at 37°C was continued for 40 min. The final reaction volume was 100 µl. Samples were analyzed by SDS-PAGE as described above, to measure the extent of cleavage. The positive control reaction of ¹²⁵I-C3(NH₃) with fH and fI did not reach complete cleavage during 40 min; thus, the data set could be used for comparison studies. Data were collected by the dissection of the dried autoradiograph and the measurement of the radioactivity of the cleavage products. The radioactivity in the 43-kDa (c) product was expressed as a percentage of the total radioactivity in each gel track. Averaged background from the negative controls was subtracted, and the data obtained were expressed as percent inhibition using the ¹²⁵I-C3(NH₃) with fH and fI control as standard (0% inhibition).

Synthetic substrates for fI

The following substrates were tested for cleavage by fI (Table I): R-AMC; GR-AMC; GGR-AMC; LGR-AMC; FGR-AMC; IEGR-AMC; FR-AMC; PFR-AMC; FSR-AMC; GPR-AMC; VPR-AMC; DPR-AMC; VLK-AMC; AAPV-AMC; and LLR-AMC. Each substrate, 25 µM (final concentration) in 50 µl of 25 mM Bicine, 0.5 mM EDTA, pH 8.25 (buffer B) with 146 mM NaCl (final concentration), was added to 3.52 µg of fI in the same buffer in a Microfluor white plate. The final reaction volume was 200 µl. The amidolytic activity of fI was measured using a microtiter plate reader (Fluoroskan; Thermo Life Sciences, Basingstoke, U.K.) by excitation at 355 nm and continuous monitoring of emission at 460 nm for 1 h or more 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 microgram of enzyme by use of a conversion factor calculated from complete substrate turnover.

The optimal pH for the AMC substrate cleavage was determined by the level of activity of fI on FGR-AMC substrate in a multiple buffer system. fI in 50 µl of 1 mM Tris-HCl, 25 mM NaCl, pH 7.4 was added to 100 µl of 20 mM acetic acid, 20 mM MES, 20 mM HEPES, 20 mM Bicine, 100 mM NaCl, pH 6.0–10.0; and 50 µl of FGR-AMC in water were added to give a final substrate concentration of 25 µM with 1 µM (88 µg/ml) fI in a final reaction volume of 200 µl (Fig. 2).

View larger version (8K): [in this window] [in a new window]   FIGURE 2. Effect of pH on FGR-AMC cleavage by fI. FGR-AMC, 50 µl in water (25 µM final concentration), was added to 50 µl of fI in 1 mM Tris, 25 mM NaCl, 0.5 mM EDTA, (1 µM; 88 µg/ml final concentration) and 100 µl of 20 mM acetic acid, 20 mM MES, 20 mM HEPES, 20 mM Bicine, 100 mM NaCl, pH 6.0–10.0. Cleavage in each sample was monitored and expressed as activity as described in Materials and Methods.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Effect of salt strength on FGR-AMC cleavage by fI. FGR-AMC, 50 µl in water (25 µM final concentration), was added to 50 µl of fI in 1 mM Tris, 0.5 mM EDTA (0.2 µM; 17.6 µg/ml final concentration) and 100 µl of 50 mM Bicine, 1.0 mM EDTA, pH 8.25, with a final NaCl concentration of 0–1000 mM. Cleavage in each sample was monitored and expressed as activity as described in Materials and Methods.

The heat stability of fI was tested in both proteolytic and amidolytic assays. Primarily a series of fI aliquots of 40 ng in 75 µl of buffer A were preheated at temperatures of 37–91°C for 30 min and tested for proteolytic activity at 37°C. The assay was stopped before complete cleavage of ¹²⁵I-C3(NH₃), and the autoradiograph produced was used for the measurement of the degree of cleavage of the -chain of ¹²⁵I-C3(NH₃) for each sample using computer software (TotalLab; Nonlinear Dynamics, Newcastle-upon-Tyne, U.K.).

FI aliquots of 3.52 µg in 50 µl of buffer B were preheated at temperatures of 37–77°C for 30 min before assay for the cleavage of the VPR-AMC substrate (25 µM final concentration). The final concentration of fI was 0.2 µM (17.6 µg/ml), and that of NaCl was 146 mM.

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.

Effect of inhibitors on fI activity

Inhibitors covering a wide spectrum of proteases were examined in the amidolytic assay. Compounds at various concentrations were preincubated for 1 h at 37°C with fI (final concentration, 0.2 µM (17.6 µg/ml)) in buffer B before the assay of cleavage of FGR-AMC (25 µM final concentration) in the same buffer with 146 mM NaCl (final concentration). The concentration of each compound used was chosen on the basis of solubility and mode of action of the compound. Compounds that caused significant inhibition were explored further in separate dose dependence measurements under conditions identical with those used for the initial screening.

Effects of C3(NH₃) and fH on fI amidolytic activity

At a final concentration of 0.1 µM (8.8 µg/ml), fI was incubated alone or in various combinations with up to 5-fold molar excess of fH, C3(NH₃) or BSA (a negative control) in 150 µl of 25 mM Bicine, 0.5 mM EDTA, 152 mM NaCl, pH 8.25 for 1 h at 37°C, before the addition of the DPR-AMC substrate (25 µM final concentration) in 50 µl of the same buffer. fH, C3(NH₃) and BSA were also tested in the absence of fI. Activity at 37°C was monitored as described above.

	Results

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fI activity on peptidyl-AMC substrates

The enzyme specificity of fI was explored by examining its catalytic capacity using the panel of 15 peptidyl-AMC substrates listed in Table I. A summary of the results obtained via spectrofluorimetry is shown in Fig. 4A. The greatest release of the AMC fluorophor was observed using the Asp-Pro-Arg derivative. Detectable enzyme activity was also observed using Val-Pro-Arg, Gly-Pro-Arg, Leu-Leu-Arg, and Phe-Gly-Arg. The Asp-Pro-Arg-AMC derivative is typically supplied as a substrate for use in determining activity of thrombin and trypsin. The Val-Pro-Arg and Gly-Pro-Arg derivatives are routinely used to assay thrombin activity, and the Phe-Gly-Arg derivative is used to detect the activity of tissue plasminogen activator. In the fI assays, cleavage was also seen with Val-Leu-Lys, Pro-Phe-Arg, Phe-Arg, Arg, Gly-Gly-Arg, Phe-Ser-Arg, and Ile-Glu-Gly-Arg substrates, although at lower levels. There was no detectable cleavage of Leu-Gly-Arg, Ala-Ala-Pro-Val, and Gly-Arg derivatives. All substrates tested, with the exception of Val-Leu-Lys-AMC and Ala-Ala-Pro-Val-AMC, had arginine at their P₁ position. Val-Leu-Lys-AMC was cleaved at a comparatively low rate, and Ala-Ala-Pro-Val was not cleaved at 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 cleavage 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 natural 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.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. A, Cleavage of peptidyl-AMC substrates by human fI. Fifteen peptidyl AMC substrates (25 µM final concentration) in 50 µl of 25 mM Bicine, 146 mM NaCl, 0.5 mM EDTA, pH 8.25, were added to 3.52 µg of fI in 150 µl of the same buffer in Microfluor white plate wells and incubated for 1 h at 37°C. Cleavage was monitored continuously by measuring emission at 460 nm, and the level of activity is shown as picomols of AMC released per minute per microgram of enzyme. Human thrombin (0.01 µg) was used as a calibration standard because it releases all AMC present within 1 h. Substrates are as defined in Table I. B, Calculation of Km and Vmax for selected substrates cleaved by fI. The values were calculated using the Lineweaver-Burk plot method. Each calculation was based on a mathematical equation derived for the best fit trend line for each set of data corresponding to a particular substrate. The concentration of the substrate ([S]) is micromolar, and the reaction velocity (v) is measured in picomols of AMC released per minute per microgram of enzyme.

The V_max values calculated for thrombin are, however, much higher in relation to these calculated for fI. For the Asp-Pro-Arg and the Val-Pro-Arg substrates, the V_max values of thrombin are 6.3 x 10⁴- and 4.6 x 10⁴-fold higher respectively than for fI. For the Phe-Gly-Arg-AMC substrate, the V_max for thrombin is 4.5 x 10³-fold higher than for fI. Thus, relative to thrombin, fI has similar affinity for these substrates, but much lower catalytic efficiency.

Investigation of effects of fH and C3(NH₃) on the amidolytic activity of fI.

To explore whether fH or C3(NH₃) affect the amidolytic activity of fI, it was decided to test the levels of the amidolytic activity of fI against the Asp-Pro-Arg-AMC substrate in the presence of either or both fH and C3(NH₃). As shown in Fig. 5, excess fH, C3(NH₃), or a mixture of both have no effect on the amidolytic activity of fI. In addition, BSA used as a negative control also has no effect, and these proteins have no intrinsic (or contaminating) amidolytic activity. For cleavage of the natural substrates C3b and C4b by fI, it has been hypothesized that protein-protein interactions among fI, fH, and substrate are required to induce formation of fI into a functionally active state. However, fH and C3(NH₃) clearly do not 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 orientation. The amidolytic activity assay is suitable for the detection and quantification of fI activity independent of cofactor proteins.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Investigation of possible effects of fH and C3(NH3) on the amidolytic activity of fI. fI at a final concentration of 0.1 µM (8.8 µg/ml) was incubated alone (0 molar excess-positive control) or in various combinations with fH, C3(NH3) or BSA (control) of up to 5x molar excess over fI, in 150 µl of 25 mM Bicine, 0.5 mM EDTA, 152 mM NaCl, pH 8.25, for 1 h at 37°C, before the addition of the DPR-AMC substrate (25 µM final concentration) in 50 µl 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.

To determine the optimal conditions in which fI exhibits the highest level of amidolytic activity, a series of assays were conducted over a range of pH values, and also NaCl concentrations. In the range of pH values examined, pH 6–10, the activity appeared to rise steadily from pH 6.0 reaching a peak at 8.25, after which there was a gradual decrease (Fig. 2). The bell-shaped curve centered around pH 8.0 is characteristic of many serine proteases.

The activity of fI showed a moderate increase with the increase of NaCl concentration between 0 and 145 mM reaching a maximum in the region of physiological salt strength. At higher salt concentrations, the activity showed a small decrease, reaching a 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 <2-fold difference in amidolytic activity between optimum and very low or very high salt strength, whereas an 6-fold difference in activity exists between those measured at pH 6.0 and pH 8.25.

In addition, the effect of divalent metal ions was also tested (Table III). Of 11 metal ions examined, consisting of 10 bivalent and 1 trivalent species, Zn²⁺, Mg²⁺, Co²⁺, Ca²⁺, Mn²⁺, Ba²⁺, and Ni²⁺ had no significant effect on the activity of fI. In contrast, Cu²⁺, Hg²⁺, Cr²⁺, and Fe³⁺ had an inhibitory effect, with Cr²⁺ and Fe³⁺ causing almost 60% loss of activity. The others led to a decrease in activity ranging from 0 to 30% loss. From the selection of metal ions tested, Zn²⁺, Cr²⁺, and Fe³⁺ were also tested in the proteolytic assay using C3(NH₃) as substrate (Table IV). Zn²⁺, which did not inhibit the activity of fI in the amidolytic assay, did inhibit the breakdown of ¹²⁵I-C3(NH₃) at concentrations of 20 or 100 µM as previously reported by Crossley and Porter (27). This is consistent with Zn²⁺ binding to fH, as reported by Sim et al. (28), but not binding to fI. However, Cr²⁺ and Fe³⁺ inhibit both the proteolytic and the amidolytic assays. The inhibitory effect of these two ions has not been tested before in the proteolytic assay. Crossley and Porter (27) had previously reported that Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, and Ni²⁺ do not affect fI cleavage of C3(NH₃) in the presence of fH.

The heat stability of fI was tested in both the proteolytic and the amidolytic assays. Fig. 6 shows the loss of enzymatic activity over an extended range of temperatures of 37–91°C. In both assays, fI loses 50% of its activity between 37 and 65°C, with >80% loss at 75°C. In neither assay was the activity completely lost at temperatures close to 80°C, indicating that the serine protease domain of fI shows a strong degree of thermal stability. In a multidomain protein, it might be expected that overall activity would be lost at the melting temperature of one particular domain, such that at one critical temperature there would be a very large loss of activity. This is not the case here given that loss of activity in the amidolytic assay increases regularly with the increase in temperature. In the more detailed analysis of the proteolytic assay, the loss of activity may occur in two stages, one between 37 and 48°C and another 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 a melting event occurring at two discrete points.

View larger version (12K): [in this window] [in a new window]   FIGURE 6. Heat stability of fI. Two sets of fI samples, one in 10 mM potassium phosphate, 0.5 mM EDTA, pH 6.2 (buffer A) and the other in 25 mM Bicine, 0.5 mM EDTA, pH 8.25 (buffer B), 145 mM NaCl were preheated at temperatures of 37–91°C for 30 min. Each set was tested independently for its activity, the first in the proteolytic assay and the second in the amidolytic assay. For the proteolytic assay, aliquots of 40 ng of fI in 75 µl of buffer A were exposed to temperatures of 37–91°C and tested for activity at 37°C. The level of activity in each sample was expressed as percent total activity lost compared with the 37°C result set as 0% loss. For the amidolytic activity assay, aliquots of 50 µl containing 0.8 µM fI in buffer B were heated at 37–77°C before testing on VPR-AMC. The final salt concentration was 145 mM NaCl. The activity data were plotted as a function of temperature.

To test the effect of inhibitors on fI, both the proteolytic and the amidolytic assays were used, the latter more extensively. In all cases an excess of inhibitor was incubated with fI for 1 h at 37°C before the addition of the substrate. Inhibitors, both natural and synthetic, were first tested in the amidolytic assay (Table V). Selected compounds from this screen were subjected to further analysis with dose dependence responses in the amidolytic assay and in the proteolytic assay for assessment of their impact on the physiological 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 activity of fI (60–100% inhibition). In contrast, PMSF, aprotinin, leupeptin, SBTI, LBTI, hirudin, and ACA showed a more moderate inhibition of activity (10–40% 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 leupeptin, antipain, BoroMPG, Pefabloc SC, suramin, and benzamidine was demonstrated (Fig. 7). Most notably, antipain and leupeptin showed strong inhibition, 80% at 25 µM, although complete inhibition was not achieved. A 50% inhibition was observed with BoroMPG, Pefabloc SC, and Suramin at concentrations of 25, 40, and 125 µM, respectively. Benzamidine reached a plateau of inhibition close to 80% at 10 mM. From all the compounds analyzed, only Pefabloc SC appeared to show complete enzyme inhibition.

View larger version (14K): [in this window] [in a new window]   FIGURE 7. Dose-dependent inhibition of fI by selected compounds as tested on the amidolytic assay. A, Leupeptin, antipain, BoroMPG, Pefabloc SC, suramin; B, Benzamidine. Different dilutions from stock preparations of each compound in 50 µl of 25 mM Bicine, 146 mM, 0.5 mM EDTA, pH 8.25, were coincubated for 1 h at 37°C with 100 µl of 0.4 µM (35.2 µg/ml) fI in the same buffer. Each mixture was transferred to 50 µl of FGR-AMC substrate (25 µM final concentration) in the same buffer in a Microfluor white plate well.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although the physiological activity of fI has been broadly characterized, detailed descriptions of the potential range of its catalytic 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 structure 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 functionally 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 substrates. This shows that the enzyme in its native state has a conformation 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 possibility that fI-cofactor interaction alters the conformation of fI to provide a secondary substrate binding site 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 µM) to C3(NH₃) varied from 50- to 250-fold molar excess, such that any competitive effect would be small.

The substrate specificity patterns of fI are dependent on the architecture of the serine protease domain of fI that aligns the desired arginyl bond to the active site area. The strong preference for Arg at P₁ (Fig. 4A) is determined by the presence of an Asp residue at position 189, located at the bottom of the specificity pocket that interacts electrostatically with positively charged residues like Arg or Lys. For the P₂ position, there is a preference for Pro. This selectivity at S₂ is under the influence of Ser²¹⁴. Surprisingly, at the natural cleavage sites, Gly, Ser, and Leu are preferred in P₂ (Pro-Ser-Arg, Leu-Leu-Arg in C3b and Thr-Gly-Arg, Arg-Gly-Arg in C4b) instead of Pro. For position P₃, little information can be gained because, in the substrates that were cleaved best, the residues at the P₃ were diverse (Table I). In C3b and C4b cleavage sites, Pro, Leu, Thr, and Arg all occur at the P₃ position. It is difficult to judge which residues are preferred in P₃, but those that can form two antiparallel strand hydrogen bonds with the Gly at position 216 (32) are likely to be the optimal.

fI and thrombin appear to have similar synthetic substrate specificities, but they cleave at very different rates of catalysis (Table II). The similar substrate specificities can be attributed to homologies within the substrate binding sites for both proteins (29, 30). Earlier studies on the specific activity of fI on C3(NH₃) in the presence of fH had reported higher cleavage velocities than the ones reported here (33). The turnover rate reported for the cleavage of the natural substrate C3(NH₃) at 37°C in physiological conditions is 900 pmol of C3b cleaved per min per µg of fI, which is significantly higher than the values described for the cleavage of tripeptides by fI. The V_max values obtained for thrombin are still considerably higher. Although the two assays are significantly different, making direct comparisons difficult, the efficiency of cleavage of the natural substrate is likely to be higher, because the conformational effects that occur are likely to contribute to increased efficiency.

Clotting proteases share ancestry with complement proteases (34, 35). The limited substrate range and low catalytic activity of fI is most likely related to its narrow activity against only two natural substrates, in the presence of cofactors. A difference between fI and thrombin is the lack of allosteric regulation of fI activity by Na⁺ ions. According to Dang and Di Cera (36), the presence of a Tyr at position 225 in thrombin supports the binding of a Na⁺ ion in a designated binding loop, whereas the replacement of Tyr²²⁵ with a Pro in fI results in no such regulation. In contrast, the proteolytic activity, which can be assessed only when a cofactor protein and a protein substrate are present, is very dependent on salt strength. The rate of reaction decreases very substantially between 10 and 150 mM (26). In addition, the pH optimum for this reaction when fH is the cofactor is low (<5.5), but when CR1 is the cofactor, the optimum pH lies between 7 and 7.5 (26). These significant differences between the proteolytic and amidolytic assays presumably reflect the weak ionic interactions fI-cofactor, fI-substrate, and substrate-cofactor (5). The interactions C3(NH₃)-fI, C3(NH₃)-fH, and fI-fH are stronger at low ionic strengths and have pH optima lower than 6 (5).

The effects of inhibitors on the amidolytic assay confirmed the serine protease nature of fI. When Crossley and Porter (27) tested a series of inhibitors on fI, they reported poor reactivity with several serine protease inhibitors. DFP, for example, did not inhibit. fI was characterized as a serine protease from sequence determination (6, 7). The activity of additional inhibitors reported in this paper is consistent with the nature of fI. As expected, bestatin, an amino- and exopeptidase inhibitor, and 1,10-phenanthroline, a metalloprotease inhibitor, did not inhibit at all, because no metals were found that regulate positively the activity of the enzyme. Pepstatin, an aspartic acid protease inhibitor, and chymostatin, a specific inhibitor of chymotrypsin, also did not inhibit. No inhibition was found with C1 inhibitor, a well-characterized serpin that inhibits C1s, C1r, MASP-1, MASP-2, plasma kallikrein, factor XIIa, and tissue plasminogen activator. The cleavage site in human C1 inhibitor has the sequence Ser-Val-Ala-Arg, which is unlikely to be compatible with the fI active site architecture. ACA, a lysine analog, does cause slight inhibition, whereas benzamidine, an arginyl analog, gave much stronger inhibition in accordance with the arginyl preference.

The moderate inhibition by SBTI and LBTI is probably due to trace contaminants in the preparations or perhaps substrate competition effects. Because these inhibitors form 1:1 complexes with their natural targets, it would be expected that if there was inhibition, at such high molar excess (250x for SBTI and LBTI), the percentage loss of activity should be 100%. This was not explored further. Hirudin gave a small effect at 1:1 molar ratio. Of the Pefabloc variants examined, Pefabloc TH is sold as a more selective inhibitor for thrombin, whereas Pefabloc Xa is relatively selective for factor Xa. Pefabloc SC was designed to cover a wider spectrum of proteases. Surprisingly, Pefabloc SC and Xa inhibited fI much better than Pefabloc TH, despite indications that fI had specificity similar to that of thrombin. This variability could be due to the presence of a Tyr at position 99 in Xa and fI, whereas thrombin has a Leu. The larger Tyr⁹⁹ residue should make the aryl binding pocket of fI smaller than that of thrombin (30). Pefabloc SC inhibits fI very effectively, whereas the other wide range serine protease inhibitor, DFP, does not inhibit fI (27).

Suramin inhibits well in both assays. It is a hexasulfonated naphthylurea with a symmetrical structure (37) that has been used for decades in the treatment of trypanosomiasis and onchocerciasis (38). It has proved useful as an antitumor agent (39) and as a potent reversible inhibitor of the complement system (40) and of C3b breakdown (41). The first reports that suramin inhibits C3b breakdown to iC3b were from Tamura and Nelson (42) and Lachmann et al. (41). It was assumed then that binding of suramin to C3b blocked the cleavage by fI, because treatment of C3b-coated cells with suramin, before exposure to fI, inhibited breakdown of C3b to iC3b (40). However, it is clear that suramin inhibits fI directly (Table V). Suramin interacts with many proteins and proteases, including trypsin (43). The similar degree of inhibition in both assays indicates that suramin does not have strong direct effects on fH or C3b. The affinity for suramin may be related to the basicity of proteinases (39): the electrostatic interactions between the negatively charged sulfonate groups of the polyanion and positively charged basic amino acids of proteases are likely to be the main interactions in the enzyme-suramin complex.

Leupeptin and antipain both inhibit fI effectively. Leupeptin has the sequence Leu-Leu-arginal which matches one of the natural cleavage sites in C3b and the synthetic substrate Leu-Leu-Arg-AMC. Antipain has an Arg-Val-arginal sequence that resembles the Arg-Gly-Arg natural cleavage site in C4b. Leupeptin has previously been described to inhibit thrombin as has aprotinin (44). The moderate inhibition of fI by aprotinin that bears a Lys-Ala-active site is consistent with the Arg preference of fI already discussed. The BoroMPG inhibitor was tested as a representative of the boropeptide thrombin inhibitors reported to cross-react with fI (29, 30). It inhibits fI potently but is of limited solubility.

Benzamidine and PMSF hardly inhibited in the proteolytic assay but were effective in the amidolytic assay. Crossley and Porter (27) also found benzamidine ineffective in the proteolytic assay. Possibly its affinity with Asp¹⁸⁹ is much lower compared with the natural substrate, and on complex formation the analog is likely removed. The multiple interactions with substrate surfaces are likely to increase the stability and enhance the interactions that result in the removal of the low molecular mass analog. Similar effects were observed for the peptide analogues antipain and BoroMPG. Similarly to benzamidine, the fI-C3(NH₃) complex formation is likely to remove the peptide analogs due to higher affinity for the natural substrate. In both tests, aprotinin was found to inhibit fI to a similar, moderate extent. Differences between the two assays may also be partly due to a pH effect. More extensive tests of affinities at different pH and salt values are required for a fuller investigation of the effects of inhibitors.

In conclusion, this work identifies means to assay fI alone without a cofactor. The novelty of the exploration of the active site of fI with substrates and inhibitors provides valuable information toward the elucidation of the configuration of the active site and the specificity regions. Mapping of certain specificity regions and characterization of the interactions within and around the active site can provide powerful information that may guide the effort for synthesis of specific inhibitor compounds that could have important medical potential. Future analysis of a three-dimensional structure will complement the current observations.

	Acknowledgments

 
We thank Dr. D. A. Mitchell for his help in composition of the manuscript.

	Footnotes

 
¹ This work was supported by the Medical Research Council (U.K.) and by a fees scholarship from the Department of Biochemistry, University of Oxford (to S.A.T.).

² Address correspondence and reprint requests to Dr. Stefanos A. Tsiftsoglou, Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. E-mail address: stefanos.tsiftsoglou{at}bioch.ox.ac.uk

³ Abbreviations used in this paper: fD, factor D; MASP, MBL-associated serine protease; fB, factor B; fI, factor I; fH, factor H; BoroMPG, Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester; DFP, diisopropylfluorophosphate; suramin, 8,8'-{carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1-phenylene)carbonylimino]}bis-1,3,5-naphthalenetrisulfonic acid; buffer A, 10 mM potassium phosphate, 0.5 mM EDTA, 0.1% Tween 20, pH 6.2; buffer B, 25 mM Bicine, 0.5 mM EDTA, pH 8.25; AMC, 7-amino-4-methylcoumarin; ACA, -aminocaproic acid; SBTI, soybean trypsin inhibitor; LBTI, lima bean trypsin inhibitor type IIL.

Received for publication March 15, 2004. Accepted for publication April 21, 2004.

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