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Novel Type of Serine Protease (Alternative Pathway/Complement Component 3 Convertase) D

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Novel Type of Serine Protease (Alternative Pathway/Complement Component 3 Convertase) D Proc. Natl. Acad. Sci. USA Vol. 77, No. 8, pp. 4923-4927, August 1980 Immunology Partial sequence of human complement component Factor B: Novel type of serine protease (alternative pathway/complement component 3 convertase) D. L. CHRISTIE, J. GAGNON, AND R. R. PORTER Medical Research Council Immunochemistry Unit, Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU, England Contributed by Rodney R. Porter, May 5,1980 ABSTRACT Factor B (a component of the alternative METHODS pathway of complement) is believed to contain the proteolytic site of the complex enzymes C3 convertase (C3bB) and C5 con- Purification of Factor B and Fragments Ba and Bb. Minor vertase (C3bnB). Conflicting results have been obtained in re- modifications were made to the procedure of Kerr (4) for the gard to the inactivation of these enzymes by diisopropyl phos- isolation of Factor B from outdated human plasma. The column phorofluoridate but it has been suggested that activated Factor of CM-Sephadex C-50 was equilibrated with 0.05 M (in place B (Factor B) is a serine protease with the active site in Bb, a of 0.1 M) sodium phosphate buffer at pH 6.0. Chromatography COOH-terminal fragment of approximately 60,000 molecular on both "aged" CNBr-activated Sepharose 4B and DEAE- weight. Partial amino acid sequence studies ofBb derived from Sepharose utilized a linear gradient of NaCl in 5 mM sodium human Factor B have shown that the NH2-terminal 40 residues barbital, pH 8.5/0.5 mM CaC12/2.0 mM MgCl2/40 mM NaCl have no homology with NH-terminal sequences of other serine proteases. However, positioning of a further 170 residues out (VBS++). of approximately 290 residues in two continuous CNBr frag- To prepare fragments Ba and Bb, Factor B (100 mg) was ments from the COOH terminus has shown that there is a strong incubated with C3 (10 mg) and Factor D (1 mg) in 50 ml of homology of sequence in this section. The active site residues VBS++ for 4 hr at 370C. The fragments were isolated by histidine, aspartic acid, and serine all are present in positions chromatography of the digest on a column (2.5 X 20 cm) of corresponding with those of typical serine proteases. It is sug- DEAE-Sepharose CL-6B in VBS++ with a 500-ml linear gra- gested that Factor B is a novel type of serine protease with a dient of NaCl from 40 to 200 mM. Factor b5was purified to the catalytic chain of molecular weight twice that of proteases previously studied and probably with a different activation stage following gel filtration on Sephadex G-75 as described by mechanism. Johnson et al. (9). Component C3 was kindly provided by R. B. and E. Sim and had been purified as described by Tack and In the alternative pathway of activation of complement, acti- Prahl (10). Prior to sequence studies, Bb was reduced and S- vated Factor B (Factor B) in association with C3b forms the [14C]carboxymethylated as described (9). complex proteases C3 convertase C3bB and C5 convertase Chemical Cleavages. CNBr fragments of Bb were prepared (C3b)nB (1, 2). On activation by Factor D, Factor B, a glyco- by the addition of a 10-fold weight excess of CNBr to the pro- protein of about 90,000 molecular weight, is split into an tein (10 mg/ml) in 70% HCOOH, and the mixture was kept in NH2-terminal fragment Ba and a COOH-terminal fragment the dark for 20 hr at 4'C. The mixture was then diluted 1:10 Bb (3-5). Ba is about 30,000 molecular weight and Bb is about with water and freeze-dried. The digest was chromatographed 60,000 molecular weight. Medicus et al. (6) reported that C3 on a column (3 X 90 cm) of Sephadex G-100 in 10% acetic acid, and C5 convertases of both pathways of activation are inhibited and 3.6-ml fractions were collected. Peptides were identified by diisopropyl phosphorofluoridate (iPr2P-F) and that, if a by their absorbance at 280 nm and by measurement of radio- radioactive reagent is used, it is found in the Bb fragment, activity. Pool CB-VI was purified on a column (1 X 30 cm) of suggesting that Factor B is a serine protease with the active DEAE-Sephadex with a 250-ml linear gradient of NH4HCO3 center in Bb. An earlier report, however, showed the alternative from 0.05 to 0.5 M. pathway CS convertase to be resistant to inhibition by iPr2P-F The cleavage of Asp-Pro bonds of Bb was performed by (7). Because the suggested catalytic peptide Bb is also about dissolving Bb (10 mg/ml) in 10% acetic acid containing 6 M twice the length of the catalytic peptides of all other serine guanidine hydrochloride adjusted to pH 2.5 with pyridine (11). proteases studied so far, it appeared that Factor B could not be The vial was flushed with nitrogen and sealed, and the mixture a typical serine protease. The amino acid sequences of many was incubated at 40°C for 96 hr with constant stirring. Products of these proteases are known and they show strong homology were separated by gel filtration on Sephadex G-100 as described with each other with many highly conserved features, notably above. in the NH2 terminus of the catalytic peptide and in the active Enzymatic Cleavages. Limited cleavage of Bb was carried site residues, suggesting that all have similar catalytic and ac- out by using clostripain. The enzyme (Institut Pasteur, 500 tivation mechanisms (8). units/mg) was activated by incubation at a concentration of 0.5 Sequence studies have therefore been made of Bb and have mg/ml in 20mM NH4HCO3, pH 8.0/10mM dithiothreitol for shown that, although the essential features of the active site 1 hr at 37"C. Unmodified Bb (1 mg/ml) in 20 mM NH4HCO3 residues of in similar po- serine proteases are present and are Abbreviations: C2, C3, and C5, second, third, and fifth components sitions relative to the COOH terminus, the NH2-terminal sec- of serum complement; iPr2P-F, diisopropyl phosphorofluoridate; tion, which is -u300 residues longer, does not contain the char- HPLC, high-pressure liquid chromatography; VBS++, 5 mM sodium acteristic NH2-terminal sequence of other serine proteases. barbital, pH 8.5/0.5 mM CaCI2/2.0 mM MgCl2/40 mM NaCl. 4923 Downloaded by guest on September 29, 2021 4924 Immunology: Christie et al. Proc. Natl. Acad. Sci. USA 77 (1980) at pH 8.0 was incubated with clostripain (enzyme/substrate Table 1. Amino acid compositions of Bb and peptides CB-IV, weight ratio, 1:200) for 1 hr at 37°C. The digest was then CB-VIa, CB-II, T-1, and AC-4 freeze-dried, reduced and alkylated as described above, and Amino acid composition, subjected to gel filtration on Sephadex G-100 equilibrated with Amino residues/100 residues 10% acetic acid. acid Bb CB-IV CB-VIa CB-II T-1 AC-4 was carried out by Cleavage at the arginyl residues of CB-II Asp 11.51 13.54 10.62 9.24 10.90 12.25 using trypsin after succinylation. Succinylation was carried out Thr 4.51 5.81 4.78 4.48 5.93 2.38 according to the procedure of Koide et al. (12). At the end of Ser 5.59 4.52 7.02 7.39 1.49 4.10 the reaction the protein was dialyzed against 0.15 M NH4HCO3 Hse 1.28 2.93 at pH 8.5. This resulted in the appearance of a precipitate which Glu 10.76 7.09 10.38 12.15 11.36 10.49 partially redissolved on dilution. Succinylated CB-II (0.75 Pro 5.09 3.00 8.85 5.54 6.60 5.58 mg/ml) was incubated with TPCK-trypsin (Worthington, 224 Gly 7.32 8.48 7.99 7.98 7.65 7.77 units/mg) in 0.15 M NH4HC03 (pH 8.5) for 2 hr at 37'C (en- Ala 4.98 8.50 2.93 5.53 4.08 3.38 zyme/substrate weight ratio, 1:75). The incubation was re- Val 9.53 10.44 7.72 8.67 5.56 10.38 peated with an additional amount of enzyme. The reaction was Cys 2.75 1.18 1.93 3.22 3.97 2.12 terminated by freeze-drying. The digest was redissolved in 2.5 Met 1.52 0.83 ml of 0.1 M NH4HCO3 and the soluble material was applied Ile 5.75 5.43 6.19 5.19 8.81 5.65 to a column (2 X 96 cm) of Sephadex G-50 (superfine) equili- Leu 8.08 8.05 3.13 8.06 9.10 9.79 brated in the same buffer. Fractions (1.5 ml) were collected and Tyr 3.92 6.66 2.50 3.50 5.84 1.62 their absorbance at 215 nm was determined. Smaller peptides Phe 2.73 1.28 3.83 3.10 5.70 were further purified by high-pressure liquid chromatography His 2.63 1.55 7.52 2.20 2.13 3.69 (HPLC) using a ,-Bondapak C18 column (Waters) and a 10 mM Lys 9.25 11.20 8.15 8.71 10.00 8.67 NH4HC03/CH3CN elution gradient as described by Water- Arg 4.08 1.99 7.39 4.31 3.46 5.61 field and Scrace (13). Compositions are based on values from 24-hr hydrolyses except for Polyacrylamide Gel Electrophoresis.
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