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Originally published In Press as doi:10.1074/jbc.M705646200 on October 5, 2007

J. Biol. Chem., Vol. 282, Issue 48, 34809-34816, November 30, 2007
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Profiling the Enzymatic Properties and Inhibition of Human Complement Factor B*Formula

Giang Thanh Le, Giovanni Abbenante, and David P. Fairlie1

From the Centre for Drug Design and Development, Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

Received for publication, July 10, 2007 , and in revised form, September 25, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Factor B (B) is a crucial component of the innate immune system, contributing to the formation of a multicomponent complement protease C3 convertase (C3bBb) that plays a vital role in triggering complement-mediated immune responses to infection and injury (1, 2). Factor B circulates in human serum in an inactive zymogen conformation (1, 2), itself consisting of a three-complement control protein (CCP) domain, a von Willebrand factor type A (vWFA)2 module, and a serine protease domain (Fig. 1). Following binding to C3b, factor B is recognized and hydrolyzed between its CCP and vWFA domains by factor D, leading to formation of the heterodimer complex C3bBb, the C3 convertase of the complement alternative pathway.

Recent crystal structures of the serine protease domain (3), the vWFA module (4), a modified Bb (5), and factor B itself (6) have revealed that factor B adopts a chymotrypsin-like fold with several novel structural motifs. The catalytic triad residues Asp551, His501, and Ser674 were in a typical active conformation found in serine proteases; however, residues that normally define the oxyanion hole produced an unusual conformation because of the inward orientation of the carbonyl group of Arg671 hydrogen-bonded to the NH group of Ser674 to form a 310 helix. This abnormal conformation is believed to be responsible for the zymogen property of factor B under physiological conditions. The vWFA domain contains an integrin-like MIDAS (metal ion-dependent adhesion site) motif that may serve as a binding site for C3b, consistent with the requirement for Mg2+ to form C3bBb from C3 and factor B.

In its native state, factor B has negligible proteolytic activity on its native substrate protein C3 at physiological pH. When factor B is bound to C3b and processed by factor D (Fig. 2), the resulting C3bBb is an efficient and specific serine protease that cleaves C3 (Km 5.9 µM, kcat 1.78 s–1) (7). However, upon irreversible dissociation, the catalytic domain Bb retains only diminished (1%) proteolytic activity (8). Despite having an inactive zymogen-like oxyanion conformation, factor B still exhibits some esterolytic activity against thiobenzyl ester substrates, including dipeptide analogue Cbz-KR-SBzl, for which the highest catalytic efficiency has been reported (kcat/Km 1370 M–1 s–1) (9). Under the same conditions, the isolated Bb domain displayed slightly improved catalytic efficiency (kcat/Km 2320 M–1s–1) due mainly to a lower Km (0.58 versus 1.19 mM). Neither a factor B-specific assay nor any other substrates have been reported to date.

Recently, our understanding about the precise role of factor B in the alternative pathway has expanded through the availability of gene-depleted mice and monoclonal antibodies (10, 11). There is now growing evidence for an essential role of factor B in activating the alternative complement pathway in models of numerous inflammatory diseases (10, 11). Studies using wild type, factor B-deficient, or C4-deficient mice in conjunction with specific anti-factor B monoclonal antibodies have demonstrated that factor B is critical for allergen-induced development of airway hyper-responsiveness and inflammation (11, 12). Factor B deficiency has been reported to ameliorate K/BxN serum transfer and collagen-induced models of rheumatoid arthritis (11, 13). As an indispensable component of the alternative pathway, factor B has also been implicated in inflammatory disorders of renal tissue injury (11, 14–16). A functionally intact alternative, but not classical, complement pathway was demonstrated to be crucial in renal ischemia-reperfusion injury, whereas both pathways are required for development of intestinal ischemia/reperfusion tissue damage (11, 17–19). Brain ischemia results in complement activation with C3 levels that are significantly higher in infarcted than in non-injured brain (20). Both C3 and neuronal ischemia are attenuated by intravenous immunoglobulin (IgG) that scavenges complement fragments (20). The alternative pathway also proved to be important in kidney inflammation such as lupus nephritis and type II membranoproliferative glomerulo-nephritis (11, 21–25). When factor B was neutralized with a specific monoclonal antibody, mice were significantly protected from complement activation and fetal loss stimulated by antiphospholipid antibody (11, 26). Human complement receptor of the immunoglobulin superfamily (CRIg), a factor B-specific inhibitor, also reversed inflammation and bone loss in two mouse models of arthritis (27).


Figure 1
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  		FIGURE 1.
Schematic representation of factor B showing components CCP1–3 (three-complement control proteins), vWFA (von Willebrand factor type A), and SP (serine protease).

 


Figure 2
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  		FIGURE 2.
Complement activation paths. Activation of the classical/lectin pathways results in assembly of C4b2a whereas activation of the alternative pathway leads to formation of C3bBb, both of which may be C3 convertases. The cleavage product C3b of either pathway can bind to factor B, generating more C3bB and amplifying the alternative pathway of complement activation.

 
To date, no specific small molecule inhibitors of factor B have been described, and only a few moderately active and non-selective inhibitors of C3/C5 convertase are known, such as FUT175 (IC50, 4 µM) (28) and the allosteric inhibitor compstatin (IC50, 12 µM) (29). The former compound (nafamostat) is in clinical use in Japan to treat acute pancreatitis and glomerulo-nephritis, but it also inhibits a wide range of targets, including many human serine proteases (30, 31). A major problem in developing inhibitors of C3 convertase has been the difficulty in readily measuring C3 convertase activity. Additionally, substrate-based analogues have so far failed to inhibit processing of C3 by C3 convertase (30–32). The availability of small molecule inhibitors of factor B and C3 convertase could be valuable for probing physiological and patho-physiological roles of factor B in vivo and for evaluating these enzymes as possible therapeutic targets for new complement-based drugs. Here we report the development of a valuable new rapid chromogenic assay for measuring factor B activity in vitro, the discovery of some important and unexpected molecular and catalytic properties of factor B for substrate processing, and the discovery of the first substrate-based inhibitor of factor B that also inhibits C3 convertase. These results provide a valuable beginning to better understanding the activity of factor B and C3 convertase, to a more robust method of screening potential inhibitors, and to the rational development of potent and selective inhibitors of C3 cleavage resulting from complement activation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Factor B, factor D, C3, and cobra venom factor were obtained from Calbiochem, or Quidel. Protein molecular weight markers for gels were purchased from Sigma. Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acids were from Novabiochem. Gel ingredients, casting apparatus, and resolving chamber (Mini-PROTEAN Tetra CellTM) were from Bio-Rad. Proteins were visualized using colloidal Coomassie Brilliant Blue staining solution. All other chemical reagents were analytical grade from Sigma.

Para-nitroanilide (pNA) Substrate Synthesis—pNA substrates were synthesized according to the general method of Abbenante et al. (33) and characterized by analytical high performance liquid chromatography and mass and NMR spectroscopy (see supplemental material).

Enzymatic Characterization and Substrate Analysis—Factor B was assayed against short peptide substrates corresponding to the relevant cleavage site in the endogenous substrate C3 but with a chromogenic pNA group in the P1'-position at the C terminus. Cleavage of pNA from the peptides by factor B produces a yellow color that enables continuous monitoring of absorbance changes at the wavelength 405 nm. The assay was conducted in a 96-well plate, with a final reaction volume of 150 µl containing factor B at concentrations of 25, 50, or 75 nM using semi-optimized conditions (100 mM glycine-HCl buffer, pH 9.5, 154 mM NaCl, 1% Me2SO). Eight different substrate concentrations, each in duplicate, were used for determining kinetic constants. After preincubation in separate wells (5 min, 37 °C), catalysis was initiated by mixing substrate with enzyme buffer solution with automatic shaking for 5 s. The optical density was measured at 405 nm every 30–60 s for 30 to 120 min (depending on activity) in a Fluostar Optima (BMG Labtech) reader, and the average change in millioptical density/min was calculated. Kinetic parameters were calculated from weighted nonlinear regression of the initial velocities as a function of the eight substrate concentrations using GraphPad Prism 4 {square} software. The parameters kcat, Km, and kcat/Km were calculated assuming Michaelis-Menten equilibrium kinetics, v = Vmax[S]/ ([S] + Km). Duplicate measurements were taken for each data point, and means ± S.E. are reported.


Figure 3
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  		FIGURE 3.
Factor B cleaves C3 at high pH. C3 (10 µg) and factor B (4 µg) were incubated at 37 °C in 32 µl of buffer at the indicated pH (154 mM NaCl in all buffers) for 3 h. a, top region of 7% gel, loading of 2.5 µg of C3/well. b, bottom region of 12% gel, loading of 5.0 µg of C3/well. Lane 1, molecular mass markers (SigmaMarker; Sigma). Lane 2, C3 showing {alpha} (112 kDa) and β (68 kDa) chains. Lane 3, factor B (top) and C3a (bottom). Lanes 4–7, C3 plus factor B (pH 7.4 (PBS), 8.3 (Tris), 9.3 (glycine), 10.2 (glycine) showing pH-dependent formation of C3a after 3 h at 37°C. Lane 8, C3 plus C3 convertase (pre-formed from cobra venom factor, factor B, and factor D in PBS). Full gels of panels a and b are available in supplemental Fig. S1, a and b, respectively.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Factor B Cleaves C3 at Alkaline pH—Factor B circulates in serum intact and is known to be inactive against its native substrate C3 at pH 7.4. We now report that factor B alone does in fact cleave C3 fairly rapidly but only at higher pH (pH > 9) and with moderate efficiency. For example, Fig. 3 shows a mini-gel analysis indicating that incubating factor B with C3 in buffers of varying pH > 8 at 37 °C for 3 h results in formation of a protein corresponding to C3a (lane 3), verified by a commercial sample ([M+H]+ 9084 Da). A band for factor B at 97 kDa remains intact at all pH values examined (lanes 4–7), but in the presence of C3 another band at ~100 kDa is also produced at pH 9.3 and 10.2 (lanes 5–7), corresponding to the cleavage product of the {alpha} chain of C3. These new protein bands are identical to those obtained (Fig. 3) from cleavage (pH 7.4, 37 °C) of C3 by C3 convertase (lane 8) generated by mixing cobra venom factor, factor B, and factor D (32). The C3 samples used (>98%) (Calbiochem) have an apparent molecular mass of 180 kDa, appearing on SDS-PAGE gels as a single band or two bands under non-reducing or reducing conditions, respectively. The ability of factor B to process C3 at alkaline pH is independent of the concentrations of Na+, Mg2+, or the metal ion scavenger EDTA (supplemental Fig. S2, a and b).


Figure 4
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  		FIGURE 4.
Hydrolysis of decapeptide Ac-ARASHLGLAR-pNA (1) by factor B or trypsin. Substrate (1 mM) was incubated with factor B (50 nM) at pH 9. 5 or trypsin (50 nM) at pH 8.0, 37 °C for the indicated time periods. The samples were filtered through a size exclusion filter with 10-kDa cut-off and analyzed by analytical high performance liquid chromatography. Identities of products were confirmed by liquid chromatography-mass spectrometry.

 
Because these results indicated that hydrolytic activity of factor B proportionally increased with pH, we also examined the ability of factor B alone to hydrolyze synthetic peptides of varying length, corresponding to the native cleavage sequence in C3, at pH 7.4 or 9.5. We found that factor B hydrolyzed the 15-residue synthetic peptide substrate AcQHARASHLGLAR{downarrow}SNL-NH2, corresponding to the native sequence at the cleavage site in C3, significantly faster at pH 9.5 than at pH 7.4. The enzyme only recognized the native cleavage site LAR{downarrow}SNL with no hydrolysis detected at the non-native Ac-QHAR{downarrow}AS site. By contrast, trypsin cleaved the same peptide at both sites (data not shown). Similar observations were made for the corresponding but shorter 10-residue peptide Ac-ARASHLGLAR-pNA (1), in which the prime side tripeptide component was replaced by the chromogenic para-nitroaniline (Fig. 4). At pH 9.5, factor B only catalyzed hydrolysis at the native cleavage site LAR{downarrow}pNA, with negligible hydrolysis detected even after 24 h at the non-native Ac-AR{downarrow}ASH site. Trypsin cleaved the same peptide at both sites at pH 8 (Fig. 4).

Development of a Chromogenic Assay for Factor B—Toward the development of a rapid chromogenic assay for monitoring factor B activity in vitro, a library of systematically shortened pNA substrates was prepared to examine effects of peptide sequence and length. For comparison, we also included Cbz-KR-pNA, which has the non-prime site identical to that of the best reported thiobenzyl ester substrate (9). Preliminary results indicated that octapeptide Ac-ASHLGLAR-pNA (3) was one of the best substrates, so we used it to optimize the conditions of the chromogenic assay described below.

Effect of pH on Enzymatic Activity—The catalytic activity of factor B against octapeptide Ac-ASHLGLAR-pNA (3) was investigated over a wide pH range, using a variety of buffers (Tris-HCl, pH 7.5–8.50; glycine-HCl, pH 9.00–10.50; piperidine-HCl, pH 10.75–11.5). The optimal pH for most rapid processing of this substrate by factor B was ~10.5 (Fig. 5). Auto-hydrolysis of this substrate was negligible at or below pH 11.5 in the absence of factor B, but the catalytic activity decreased abruptly above pH 10.5, presumably due to denaturation of factor B. The effect of pH on the catalytic activity of factor B was independent of Na+ (154 mM NaCl, Fig. 5; 0 mM NaCl, supplemental Fig. S3).


Figure 5
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  		FIGURE 5.
Dependence on pH of hydrolysis of Ac-ASHLGLAR-pNA (3) by factor B. Ac-ASHLGLAR-pNA (1 mM) was incubated with 75 nM factor B at pH 7.5, 8.5, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, 11.0, 11.25, and 11.5. Initial velocity (µM/s) was calculated as amount of free pNA released from increase in millioptical density/s (at 405 nm). All buffers contained 154 mM NaCl.

 
Effect of Divalent Cations, Ion Strength, and Other Additives—Effects of diluents were probed at pH 9.5. Although the vWFA domain of factor B contains an integrin-like MIDAS (metal ion-dependent adhesion site) motif that could bind divalent cations, neither calcium (10 mM), magnesium (10 mM), nor the divalent cation-sequestering agent EDTA (10 mM) detectably influenced the activity of factor B in vitro. Similarly, neither variation of ionic strength (NaCl 0–250 mM) nor addition of bovine serum albumin (1%), polyethylene glycol (1%), or glycerol (10%) had any effect on the substrate-processing ability of factor B. Under the semi-optimal conditions (pH 9.5, 100 mM glycine-HCl buffer, 154 mM NaCl), we found that factor B exhibited classic Michaelis-Menten kinetics against the pNA substrate 3, with kcat 10.19 ± 0.19 s–1, Km 2.19 ± 0.08 mM, and kcat/Km 4666 ± 253 M–1s–1 (Fig. 6).

Effects of Substrate Length—A number of pNA peptide substrates were synthesized, ranging from 4 to 10 amino acids corresponding to the native cleavage sequence of C3. Because Cbz-KR-SBzl has been reported to be the best thioester substrate for factor B (9), its corresponding pNA analogue, Cbz-KR-pNA, was also prepared for comparison.

Among eight putative substrates examined (Fig. 7), the 8- and 7-residue pNA peptides (3 and 4) proved to be the best substrates, with initial velocity of 0.17–0.19 µM {square}s–1 (50 nM factor B, pH 9.5, (substrate) = 1 mM). The Cbz-KR-pNA substrate displayed >10-fold lower initial velocity under identical conditions. Km and kcat values for selected substrates are recorded in Table 1. Heptapeptide substrate 4 (Km 8.4 mM, kcat 1 s–1, kcat/Km 8315) was processed most efficiently by factor B. When this substrate was examined using factor B obtained from a different supplier (Quidel), comparable catalytic efficiency was observed (Km 1.15 ± 0.21 mM, kcat 9.1 ± 0.6 s–1, kcat/Km 8258 ± 2070 M–1s–1).


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  		TABLE 1
Kinetic parameters for pNA peptide substrates

Km and kcat were determined using initial velocities for selected 7-10-residue substrates at concentrations 0.2, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mM.

 


Figure 6
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  		FIGURE 6.
Hydrolysis of octapeptide substrate (3). Ac-ASHLGLAR-pNA (3) for different concentrations of factor B enzyme (25, 50, 75 nM) and pNA substrate (0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 3.0 mM) at pH 9.5, 37 °C. Initial velocity (µM/s) was calculated as amount of free pNA released from increase in millioptical density/s (at 405 nm).

 


Figure 7
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  		FIGURE 7.
Effect of substrate length on factor B catalysis. Substrates are AcARASHLGLAR-pNA (1, 10-mer), Ac-RASHLGLAR-pNA (2, 9-mer), Ac-ASHLGLAR-pNA (3, 8-mer), Ac-SHLGLAR-pNA (4, 7-mer), Ac-HLGLAR-pNA (6-mer), Ac-LGLAR-pNA (5-mer), Ac-GLAR-pNA (4-mer), and Cbz-KR-pNA. Substrates (1 mM) were incubated with 50 nM factor B in glycine-HCl buffer, pH 9.5, at 37 °C. Change in optical density at 405 nm in the first 5–10 min was used to calculate the initial velocities.

 
Effect of the 7th Residue in Substrates—The 7th residue serine in the heptapeptide substrate Ac-SHLGLAR-pNA (4) was varied to probe substrate requirements at this position. Large hydrophobic or negatively charged side chains at this position significantly decreased hydrolysis by factor B (Fig. 8). Of the small number of heptapeptide substrates investigated (Fig. 8), compound 4 with Ser at position 7 proved to be the best. A serine mimetic, isoxazole-5-carbonyl, was poorly tolerated, while small side chains (Ala) or positively charged moieties (Lys, Dapa (diaminopropionic acid)) caused small decreases in initial velocities (Fig. 8).


Figure 8
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  		FIGURE 8.
Initial velocities for factor B processing of heptapeptide-pNA substrates. Substrates varied in the 7th residue: Ac-AHLGLAR-pNA (5, Ala), Ac-LHLGLAR-pNA (Leu), Ac-FHLGLAR-pNA (Phe), Ac-DHLGLAR-pNA (Asp), Ac-KHLGLAR-pNA (6, Lys), Ac-Isox-HLGLAR-pNA (isoxazole-5-carboxylic acid, Isox), Ac-Dapa-HLGLAR-pNA (L-diaminopropionic acid, Dapa), Ac-SHLGLAR-pNA (4, Ser). Substrates (1 mM) were incubated with 50 nM factor B in glycine-HCl buffer, pH 9.5, at 37 °C. Change in optical density at 405 nm in the first 5–10 min was used to calculate initial velocities.

 


Figure 9
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  		FIGURE 9.
Dose-dependent inhibition of factor B by compound 7. Conditions were 100 mM glycine-HCl buffer, pH 9.5, 37 °C, 50 nM factor B, 0.5 mM pNA substrate 4, eleven concentrations (from ~1 µM to 1 m M) of 7. Positive control was buffer alone (not shown).

 
Substrate-based Inhibitor of Factor B and C3 Convertase—To convert substrate 4 to a putative inhibitor, the pNA moiety was replaced by an aldehyde group to furnish compound 7 (AcSHLGLAR-H). This compound proved to be a moderately potent inhibitor of factor B (IC50 19 µM, Fig. 9), blocking the cleavage of C3 in a concentration-dependent manner under the same assay conditions (Fig. 11, supplemental Fig. S4, a and b). In this inhibitor, the aldehyde functional group is capable of forming a reversible covalent bond with the catalytic Ser side chain, resulting in a tetrahedral configuration that mimics the transition state.

Compound 7 is a reversible and competitive inhibitor of factor B, since increasing concentrations of substrate 4 overcame the inhibition of factor B caused by 100 µM 7 (Fig. 10). Cleavage of the native substrate C3 to C3a and C3b by factor B at pH 10.2 was also inhibited by aldehyde 7 in a concentration-dependent manner (Fig. 11). Because factor B also provides the crucial catalytic unit within C3 convertase of the alternative pathway, compound 7 was also examined for inhibition of C3 convertase. Cleavage of C3 by C3 convertase generated either from a combination of cobra venom factor, factor B, and factor D (CVF.Bb; Fig. 12 and supplemental Fig. S5, a and b) or from human C3, factor B, and factor D (hC3bBb, supplemental Fig. 6, a and b) in the presence of Mg2+ was indeed inhibited by aldehyde 7 in a dose-dependent manner. Under the same conditions, general serine protease inhibitors leupeptin or phenylmethylsulfonyl fluoride at 1-mM concentrations did not inhibit C3 cleavage (supplemental Fig. S6, a and b).


Figure 10
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  		FIGURE 10.
Effect of substrate concentration on inhibition by 7. Substrate 4 at concentrations of 0.5, 2.5, 5, or 10 mM was incubated with 50 nM factor B in the presence of 100 µM inhibitor 7, 100 mM glycine-HCl buffer, pH 9.5, at 37 °C. Increases in absorbance were monitored at {lambda} = 405 nm.

 


Figure 11
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  		FIGURE 11.
Compound 7 inhibits cleavage of C3 by factor B in a concentration-dependent manner. C3 (10 µg) and factor B (4 µg) were incubated at 37 °C in 32 µl of 100 mM glycine-HCl buffer, pH 10.2, with various concentrations of inhibitor 7. a, top region of 7% gel, loading of 2.5 µg of C3/well. b, bottom region of 12% gel, loading of 5.0 µg of C3/well. Lane 1, molecular mass markers (SigmaMarker; Sigma). Lane 2, C3a. Lane 3, C3 showing {alpha} (112 kDa) and β (68 kDa) chains. Lane 4, factor B. Lanes 5–9, C3, factor B plus buffer (lane 5) or inhibitor (lanes 6–9, 0.01, 0.1, 0.25, 0.5 mM compound 7) showing inhibition of C3 cleavage to C3a after 3 h at 37 °C. Full gels of panels a and b are available in supplemental Fig. S4, a and b, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Factor B is currently considered to be an inactive zymogen serine protease involved in the alternative pathway of complement activation (1, 2). Recent studies using knock-out mice and a specific antibody have demonstrated important contributions from factor B to a number of inflammatory disorders (10, 11), such as airway hyper-responsiveness (12), lupus nephritis (34), and antiphospholipid syndrome (26, 35), and have provided insights to the role of factor B in complement activation via the alternative pathway. The new data from those studies suggest that factor B may be a potential therapeutic target in its own right. Moreover, selective inhibitors of factor B (none is known) could potentially be valuable new molecular probes for investigating the biology of factor B and its homologue C3 convertase, as well as providing clues to the development of novel therapeutics that might regulate the alternative pathway of complement activation (10, 11). To date, only a couple of weakly potent and non-selective inhibitors of C3 convertase are known (28, 29), but they are not substrate analogues and have been of little use for understanding C3 cleavage relevant to complement activation. No protease inhibitors for factor B are known, and there is no chromogenic assay available for monitoring enzyme activity for either factor B or C3 convertase in vitro.


Figure 12
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  		FIGURE 12.
Compound 7 inhibits cleavage of C3 by C3 convertase (CVF.Bb) in a concentration-dependent manner. a, top region of 7% gel, loading of 2.5 µg of C3/well. b, bottom region of 12% gel, loading of 5.0 µg of C3/well. C3 convertase was generated by incubating 12 µg of cobra venom factor, 8 µg of factor B, and 0.2 µg of factor D in 100 µl of PBS, pH 7.4, containing 1 mM MgCl2 for 1 h at 37°C, and then 100 µl of PBS, pH 7.4, containing 20 mM EDTA was added. C3 (10 µg) and the C3 convertase (6 µl) were incubated at 37 °C for 30 min in final 32 µl of PBS, pH 7.4, with buffer or various inhibitor concentrations. Lane 1, molecular mass markers (SigmaMarker; Sigma). Lane 2, C3 showing {alpha} (112 kDa) and β (68 kDa) chains. Lane 3, C3a. Lanes 4–8, C3, C3 convertase plus buffer (lane 4) or inhibitor (lanes 5–8, 0.01, 0.1, 0.25, 0.5 mM) showing inhibition of C3a formation after 37 °C for 30 min. Full gels of panels a and b are available in supplemental Fig. S5, a and b, respectively.

 
Factor B is known to be inactive against its native substrate C3 at pH 7.4 (1, 2), and this may be the reason why factor B function has not been heavily studied. Experiments reported here have shown that factor B alone can hydrolyze C3 under mild to strong alkaline conditions. Importantly, the cleavage products from factor B-mediated hydrolysis of C3 at pH 10.2 are identical to those produced by cobra venom factor-containing C3 convertase at pH 7.4, as demonstrated by gel analysis (Fig. 3). The significance of these new findings of pH-induced activation of factor B may be important in understanding catalysis by factor B. The structure of the active conformation of factor B remains elusive, as it is stable only in complex with C3b or the C3b surrogate, cobra venom factor. On the basis of recent NMR and x-ray crystal structures for an inactive state of factor B, it has been proposed that activation of factor B likely proceeds through multiple conformational changes (1, 2, 6, 36) induced by binding to C3b and then through cleavage by factor D (1, 2, 6, 36).

On the basis of our results it is conceivable that, at alkaline pH, factor B changes its protonation state and adopts an active conformation that can more effectively recognize and cleave C3. Other serine proteases are known to be activated under alkaline conditions. For example, NS2B-NS3 serine proteases from flaviviruses (e.g. West Nile, dengue, yellow fever) (37–42) become substantially active in vitro only at alkaline pH (≤10.5). Those enzymes, like factor B, are thought to interact with other protein domains and to deposit on membrane surfaces under native conditions for proteolytic processing. Human kallikrein 6 has been reported to exhibit significantly higher catalytic activity at pH 9.0 than at pH 7.5 (43); the basis for such pH dependence is not well understood at present.

The catalytic activity of factor B against small pNA substrates was also found here to exhibit a strong pH dependence. The hydrolysis of pNA substrates by factor B followed classic Michaelis-Menten kinetics. The magnitude of kinetic parameters (Table 1) for our pNA substrates supports the idea that factor B adopts an active conformation at higher pH, since all substrates are processed quite fast kcat (>8 s–1), comparable with the catalytic hydrolysis of C3 by C3 convertase (kcat 1.78 s–1). On the other hand, kcat/Km values for the pNA substrates are only modest (Table 1), due to a high Km (low mM). This suggests a much lower substrate affinity for the pNA substrates than for C3 (Km 5.9 µM for C3 processing by C3 convertase).

Strikingly, factor B prefers longer substrates than other serine proteases. We found that pNA substrates with short peptide sequences (<7 residues) were very poor substrates for factor B, unlike other serine proteases that tend to recognize shorter segments of polypeptide substrates and inhibitor analogues (30, 44, 45). Aminomethyl courmarin substrates of ≤6 residues have been reported not to be processed effectively by C3 convertase (32). Substrates with 7 or 8 residues, in contrast to shorter peptides, were found here to be readily processed by factor B. Trypsin and thrombin, which recognize shorter substrate sequences, still process the heptapeptide AcSHLGLAR-pNA and are inhibited by the corresponding AcSHLGLAR-aldehyde (supplemental Fig. S8, a and b). However, trypsin can process AcARASHLGLARSNL-NH2, corresponding to the native sequence at the cleavage site in C3, at both the AR{downarrow}A and AR{downarrow}S sites, whereas factor B only cleaved at the AR{downarrow}S site and only effectively in sequences with 7 or more residues on the N-terminal side. The crystal structure of factor B in its inactive state (3, 6) shows shallow putative binding pockets at S1-S6 in the enzyme, consistent with the need for some additional binding residues attached to hexapeptide substrates. However, the unusual requirement of small P7 (Ser) and P8 (Ala) residues for enhancement of substrate processing was surprising. Using NMR spectroscopy, we did not detect any conformational changes in water going from 6- to 7-residue peptides, which were both random coil structures (3JNH-CH{alpha} coupling constants for 6-mer were 6.8, 5.8, 7.5, 5.9, 6.6, 6.8 Hz and for 7-mer were 6.7, 8.0, 5.9, 7.2, 5.9, 6.1 Hz), so it would appear that the substrate extensions cause higher receptor affinity (note lower Km values) because of their side chains rather than a conformational change to the substrate.

The substrate-processing results may reflect that enzymatic processing of C3 is only slightly dependent on affinity or selectivity for substrate residues immediately surrounding the cleavage site. Indeed, the sequence of C3 at the cleavage site is not greatly different from that of substrates for other serine proteases. Instead, the selectivity for processing of C3 during complement activation is probably heavily influenced by the multiple domains of C3 convertase, evolved for selective recognition of this particular substrate under different complement activation conditions produced by different environmental stimuli. It has been estimated that ~40% of proteases are multidomain enzymes (46), and many of those appear to use the appended domains for selective substrate recognition.

It has been proposed that C3 binds C3 convertase through several docking sites on the C3b component of C3bBb complex, which in turn interacts with Bb to induce the active conformation (5). In a simple model of binding between C3 and Bb, the positively charged loop 2 of Bb could possibly interact with the negative charges on the anchor region and/or MG8 of C3 (47). The proposed interactions may confer the relatively high implied affinity of C3 (Km 5.9 µM) for the C3bBb complex, and thus only C3 convertase (but not Bb) can bind to C3 efficiently for hydrolysis to occur. Consistent with this hypothesis, Bb requires the presence of divalent cations (Mg2+ or Ni2+) to cleave C3, although with only 1% activity of factor B (8).

Until now, rational design of substrate-based C3 convertase inhibitors such as {alpha}-ketoheterocycles has reportedly failed (32), possibly because of the limitation of low affinity of the quite short peptide sequences examined to date. Despite the low affinity of substrate 4 for factor B, replacing the pNA moiety with an aldehyde C-terminal cap afforded a moderately active factor B inhibitor 7 (IC50 19 µM). This compound also inhibits the cleavage of C3 at high pH by factor B or at pH 7.4 by C3 convertase (generated from cobra venom factor, factor B, and factor D or from human C3, factor B, and factor D) in a concentration-dependent manner (Figs. 11 and 12, and supplemental Figs. S4-S6). Furthermore, aldehyde 7 is able to inhibit downstream formation of membrane attack complex to a comparable extent via the alternative pathway (supplemental Fig. S7).

Access to the active conformation of factor B could be quite valuable for better understanding of the molecular mechanism by which C3 convertase carries out its catalytic function and for devising more effective therapeutic strategies to intervene in prolonged complement activation. The novel results presented above suggest that factor B may adopt an active enzyme conformation at alkaline pH and, in complex with a reversible substrate-based aldehyde inhibitor, could potentially present the first opportunity to obtain crystal structures for factor B and C3 convertase in their catalytically active forms. The chromogenic assay reported here is quite robust and allows for rapid screening of compounds as putative inhibitors of factor B and C3 convertase. Compound 7, designed and evaluated using this assay, inhibits cleavage of C3 by both factor B and C3 convertase in a concentration-dependent manner. This indicates that the inhibitor is competitive and reversible and thus directed at the catalytic substrate-binding active site of both enzymes. These results for this new assay, developed for factor B but also validated here for native and synthetic forms of C3 convertase from the alternative pathway, suggest that potent substrate-based inhibitors of these serine proteases may now be achievable. Development of such inhibitors into even more potent, selective, and more drug-like compounds may facilitate rational intervention in the alternative pathway of complement activation and the blockade of membrane attack complex formation.


   FOOTNOTES
 
* This work was supported in part by the National Health and Medical Research Council of Australia and by an Australian Research Council fellowship (to D. P. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S9. Back

1 Recipient of an Australian Research Council fellowship. To whom correspondence should be addressed. Fax: 61-733462990; E-mail: d.fairlie{at}imb.uq.edu.au.

2 The abbreviations used are: vWFA, von Willebrand factor type A; pNA, para-nitroanilide; PBS, phosphate-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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