Biochem. J. (1980) 191, 173-182Printed in Great Britain

Purification of the human complement control protein C3b inactivator

L. Gail CROSSLEY and Rodney R. PORTERDepartment ofBiochemistry, University ofOxford, South Parks Road, Oxford OX] 3QU, U.K.

(Received 18 February 1980/Accepted 28 May 1980)

An alternative method of isolation from human plasma is described for C3b inactivator,C3bINA, the proteinase that in conjunction with either,lH or C4b-binding protein willhydrolyse respectively C3b or C4b, the activation products of the third, C3, and fourth,C4, components of complement. The purification is by chromatography of plasma oncolumns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite andSephacryl S-200. The yield of C3bINA (6mg from 500ml of plasma) is severalfoldhigher than in previously described methods. The sensitivity of the assay for C3bINAhas been increased by including optimal amounts of ,61H, and it was observed that fllHwas essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution orbound to a cell surface. Native C3 is not hydrolysed by C3bINA+fllH, but thehaemolytically inactive form that appears on prolonged storage at 40C or on freezingand thawing is hydrolysed and gives fragments of the a-chain of 75000 and 43000apparent mol.wt. As the a'-chain of C3b, which has lost an N-terminal peptide C3a,gives fragments of 67000 and 43000 apparent mol.wt. when incubated withC3bINA + 1PIH, this suggests that the larger fragment is N-terminal and the smaller oneC-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clearclassification of the type of proteinase to which this enzyme belongs has been obtained.

When complement is activated, by either theclassical or the alternative pathway, the com-

ponents C3b and C4b are formed by proteolyticcleavage of C3 and C4. The products C3b andprobably C4b are important, not only in continuingthe sequence of events leading to cytolysis, but alsoin acting as opsonins with other major biologicalroles. Regulation of these functions of C3b and C4bis achieved via the C3b inactivator (C3bINA),which apparently acts as a proteinase to cleave thea'-chains of C3b and C4b, in conjunction with twocofactors, ,16H and C4b-binding protein respec-tively. The functions associated with C3b and C4bare lost as a result of the action of C3bINA (forreview see Porter & Reid, 1979).

In the case of C3b, C3bINA cleaves a singlepeptide bond in the a '-chain to produce two

Abbreviations used: the nomenclature of complementcomponents and control proteins is that recommended bythe World Health Organisation (1968); activatedcomponents are indicated by an overbar, e.g. CT, or bythe activation product, e.g. C3b or C4b; C3b inactivatoris C3bINA; IgG, immunoglobulin G; IgM, immuno-globulin M; iPr2P-F, di-isopropyl phosphorofluoridate;DGV buffer, dextrose/saline/veronal/gelatin buffer; GVbuffer, saline/veronal/gelatin buffer; QAE-Sephadex,quaternary aminoethyl-Sephadex.

Vol. 191

fragments of mol.wts. 67000 and 43000, both ofwhich are disulphide-linked to the fl-chain (Pang-burn et al., 1977).C3bINA has been purified to homogeneity and

shown to have a structure of two covalently linkedchains of mol.wts. 50000 and 38000, both of whichcontain carbohydrate, as detected by the periodicacid/Schiff stain (Pangburn et al., 1977). Multiplebands between pH 5.7 and 6.1 are observed on

isoelectric focusing (Fearon, 1977). C3bINA hasbeen reported to resist denaturation at pH 2.2 or by2M-guanidine (Pangburn et al., 1977). The lowconcentration of C3bINA in serum has resulted inonly small amounts of highly purified C3bINA beingisolated.The present work reports a method employing

mild isolation techniques with precautions againstproteolysis for the purification of C3bINA, with a

substantially higher yield than has previously beenreported. Further information on the properties ofC3bINA and the cleavage products formed from C3derivatives has been obtained.

Materials and methods

MaterialsOutdated human plasma was obtained from the

0306-3275/80/100173-10$01.50/1 ©D 1980 The Biochemical Society

173

L. G. Crossley and R. R. Porter

Churchill Hospital, Oxford, U.K. Chemicals wereobtained as follows: QAE-Sephadex A-50,Sepharose 4B, Sephacryl S-200, CM (carboxy-methyl)-Sephadex C-50 and Sephadex G-75,Pharmacia Fine Chemicals, Uppsala, Sweden;hydroxyapatite (DNA-grade Bio-Gel HTP), Bio-Rad Laboratories, Bromley, Kent, U.K.; wheat-germ, local grocers and N-acetyl-D-glucosamine,Sigmna Chemical Co., Poole, Dorset, U.K.; anti-(human C3bINA) (goat) serum and anti-(humantransferrin) serum (goat), Flow Laboratories, Irvine,Ayrshire, Scotland, U.K.; anti-[human (IgG + IgM)]serum (goat), Behring Diagnostics, Marburg,Germany. The sources of other reagents have beendescribed previously (Gigli et al., 1976, 1977).

Complement componentsComponent C2 and factor B were purified by the

method of Kerr & Porter (1978) as modified by Kerr(1979). C3 was isolated by the procedure of Tack &Prahl (1976). JJlH was obtained as a by-product ofthe C3 preparation, being eluted between IgM andC3/C5 during gel filtration on Sepharose 6B, andwas purified to homogeneity by chromatography onhydroxyapatite. Factor D was purified by Sepha-dex G-75 chromatography of a fraction eluted with0.4M-sodium phosphate buffer, pH 6.0, from CM-Sephadex C-50 during the procedure for the puri-fication of C2 (Kerr & Porter, 1978; Johnson et al.,1980).

C3b was prepared by incubation of C3(5.8mg) with 150,g of factor B and 20,ug of factorD in 3.2 ml of 0.15 M-NaCl/25 mM-imidazole buffer,pH 7.0, containing 5mM-MgCl2 and 2mM-CaCl2, at370C for 30min. Polyacrylamide-gel electro-phoresis of samples of the incubation mixture takenat 5min intervals indicated that conversion of C3into C3b was complete after 10min, and less than1% of the C3 haemolytic activity (Tack & Prahl,1976) remained at the end of the 30-min incubationperiod. The incubation mixture was dialysed against0.1M-sodium phosphate buffer, pH 6.0, and appliedto a column (1.5 cm x 5 cm) of CM-Sephadex equili-brated with the same buffer. C3b was not adsorbedon the gel, and was separated from inactive C3,factor B and D, which were adsorbed and eluted onthe addition of 0.5 M-NaCl to the buffer. The C3bpreparation was homogeneous on polyacrylamide-gel electrophoresis.

Preparation ofL-lysine-Sepharose 4BL-Lysine-Sepharose 4B was prepared by the

method of Deutsch & Mertz (1970). The incor-poration of lysine was determined by amino acidanalysis of a washed and dried sample to be5,umol/ml of packed Sepharose.

Plasminogen adsorbed on L-lysine-Sepharose

from plasma (see below) was eluted with 0.2 M-e-aminohexanoic acid in 0.3 M-potassium phosphatebuffer, pH 7.0, and the gel was washed extensivelyand stored in water containing 0.02% NaN3.

Preparation of N-acetyl-D-glucosamine-Sepharose4B

Oxirane-Sepharose 4B was prepared by themethod of Sundberg & Porath (1974) andimmediately coupled with N-acetyl-D-glucosamineas described by Vretblad (1976). The gel waswashed successively with water, 0.1 M-sodium bor-ate buffer, pH 8.0, 0.1 M-sodium acetate buffer,pH 4.0, and water and finally equilibrated in acolumn with 50mM-sodium cacodylate buffer,pH7.2.

Preparation of wheat-germ agglutininWheat-germ agglutinin was purified by modi-

fication of the procedure of Shaper et al. (1973).Wheat-germ (780 g) was washed on a Buchnerfunnel with Whatman no. 4 paper with 4 litres ofn-hexane and dried overnight. The defatted wheat-germ was ground in a Waring blender in several lotsfor 3 x 15 s each. The dry ground wheat-germ wassuspended in 5 litres of water at 40C, stirred for 2hand centrifuged at 10OOg for 30min at 40C. Thesupernatant was made 55% saturated with(NH4)2SO4 (33 g/100 ml) and stirred at 40C for 2h,then centrifuged at 8000g for 30min at 40C. Theprecipitate was suspended in 800ml of ice-cold waterand centrifuged as before. The resulting supernatantwas heated in a water bath at 63 0C for 15min andcentrifuged as before to yield a clear goldensupernatant.The extract was applied to a column

(2.2cm x 15 cm) of N-acetyl-D-glucosamine-Sephar-ose 4B equilibrated with 50mM-sodium cacodylatebuffer, pH 7.2, at 35ml/h. The column was washedwith 600ml of buffer until the A280 of the eluate hadfallen to 0.06, after which the agglutinin remainingadsorbed on the gel was eluted with buffer con-taining 100mg of N-acetyl-D-glucosamine/ml. Thefractions containing the agglutinin were combined.The recovery was 27mg of agglutinin/lOOg ofwheat-germ, on the basis of A " = 14.3 (Nagata &Burger, 1974). Several preparations of wheat-germagglutinin were combined, precipitated with(NH4)2SO4 (40g/100ml) and redissolved in theminimal volume of 50mM-sodium cacodylate buffer,pH 7.2.

After each use, the N-acetyl-D-glucosamine-Sepharose 4B was washed successively on asintered-glass funnel with water, 6 M-urea containing50mM-EDTA, pH7.0, water, 30% (w/v) ethanoland water and stored in water containing 0.02%NaN3.

1980

174

Human C3b inactivator

Preparation of wheat-germ agglutinin-Sepharose4B

Sepharose 4B (lOOml) was activated with lOgof CNBr by the procedure of Cuatrecasas et al.(1968), the pH being maintained at 11 with4 M-NaOH for 8 min. The Sepharose was thenwashed on a sintered-glass funnel with 2 litres ofcold 0.1 M-NaHCO3, and the moist Sepharose wastransferred to a beaker containing 100 ml of 0.15 M-NaCl/O. 1M-NaHCO3, pH 8.3. A concentrated solu-tion of wheat-germ agglutinin (500mg in 40ml),dialysed against 0.15 M-NaCI/O. 1 M-NaHCO3,pH 8.3, containing 0.1 M-N-acetyl-D-glucosaminewas added and the mixture was stirred at 200C for3 h. The wheat-germ agglutinin-Sepharose waswashed extensively with 0.15 M-NaCl/O. 1 M-NaCO3,pH8.3, and the same buffer containing 100mg ofN-acetyl-D-glucosamine/ml, and stored at 40C in0.2 M-NaCI/50 mM-sodium phosphate buffer, pH 7.0,containing 0.02% NaN3.

Buffers used in haemolytic assay ofC3bINABuffers of the following compositions were used:

GV buffer, iso-osmotic veronal buffer, pH 7.5,containing 0.1% gelatin (Nelson et al., 1966);EDTA/DGV buffer, pH6.5, 5% (w/v) glucose inwater/GV buffer/O. 1 M-EDTA, pH 7.5 (15: 5: 2, byvol.) adjusted to pH 6.5 with 1 M-HCl; DGV buffer,equal volumes of GV buffer and 5% (w/v) glucose;DGV2+ buffer, DGV buffer containing 0.15mM-CaCl2 and 0.5mM-MgCI2; EDTA/GV buffer, GVbuffer/O. 1 M-EDTA, pH 7.5 (1:9, v/v).

Preparation ofEA C43 cellsEAC43 cells were prepared by the method of C.

Parkes, R. G. DiScipio & M. A. Kerr (personalcommunication) as follows: Sheep erythrocytessensitized by antibody and components Cl and C4(EAC 14) (Borsos & Rapp, 1967) were incubated inDGV2+ buffer (1 x 108cells/ml) with humancomponent C2 (0.4,ug/ml) and component C3(5,ug/ml) for 5min at 300C, then chilled on ice for0min. The cells were then centrifuged at lOOOg for10min at 40C and resuspended in EDTA/GVbuffer. This latter step was repeated, and the mixturewas incubated for 15min at 370C, centrifuged,resuspended in EDTA/GV buffer and incubated fora further 15 min at 37°C. The resulting EAC43 cellswere washed twice with DGV2+ buffer and stored at40C.

Assay ofC3bINAC3bINA activity was assayed by modification of

the method of Whaley et al. (1976). EAC43 cells inDGV2+ buffer (1 x 108/ml) were pelleted by centri-fugation at lOOOg for 10min, washed twice byresuspension to the same concentration of cells in

Vol. 191

EDTA/DGV buffer, pH 6.5, and finally resus-pended in EDTA/DGV buffer, pH 6.5, containinglO,ug of ?lH/ml immediately before use. Dilutions(0.1 ml) of C3bINA in EDTA/DGV buffer, pH 6.5,were incubated at 37°C for 30min with 0.1 ml of thewashed EAC43 cells (1 x 108/ml). Ice-cold DGVbuffer (0.5ml) was added to stop the reaction, andthe cells were washed twice with ice-cold DGV bufferand once with ice-cold DGV2+ buffer. The pelletedcells were resuspended in DGV2+ buffer (0.3 ml)containing factors B (7,ug/ml) and D (0.4,ug/ml) andincubated at 300C for 30min. The amounts ofcomponents B and D were predetermined to givemaximum lysis of EAC43 cells in the absence ofC3bINA in the first incubation mixture. Afteraddition of 0.3 ml of guinea-pig serum diluted1:30 with 0.04M-EDTA in GV buffer and furtherincubation at 370C for 1h, 1ml of ice-cold 0.15M-NaCl was added and the mixture centrifuged atlOOOg for 10min. The degree of lysis was measuredfrom the A410 of the supernatant and expressed as(Z') haemolytic units, 1 unit being the reciprocal ofthe dilution of C3bINA that gives Z' = 1, whereZ' = In (1-% inactivation) (Whaley et al., 1976).

Preparation ofC3bINAAll procedures were performed at 40C and all

buffers contained 0.02% NaN3. Tris buffers wereprepared by dilution of 1 M-Tris adjusted to thestated pH with HCl at 200C. Each of the columnswas used repeatedly after washing as describedwithout repacking of the column, with the exceptionof the QAE-Sephadex resin, which was regener-ated by washing successively with 0.1 M-HCl, water,0.1 M-NaOH and water.

Plasma (500 ml), to which 0.5 ml of 2.5 M-iPr2P-Fin propan-2-ol was added, was clarified by centri-fugation at lOOOOg for 30min. The supernatant wasapplied to a column (5cm x 15cm) of lysine-Sepharose equilibrated with 0.1 M-potassium phos-phate buffer, pH 7.0, containing 0.15 M-NaCl and15mM-EDTA at a flow rate of 200ml/h. Thecolumn was washed with buffer, and the A280 of theeluate was monitored and the non-absorbed proteineluted after the void volume was collected until theA280 of the eluate was less than 1.0. The plasmino-gen-depleted protein pool was made 2.5 mm withrespect to iPr2P-F and dialysed against three 3-litrechanges of 60nmM-NaCl/20mM-Tris/HCl buffer,pH 7.8. The non-diffusible material was passedthrough a column (7 cm x 36 cm) of QAE-Sephadexequilibrated with the dialysis buffer at a flow rate of300ml/h. Fractions (25ml) were collected and theabsorption at 280nm of the eluate was recorded.After washing of the column with 1.5 columnvolumes of dialysis buffer, a linear gradient (2 x 2litres) of the buffer containing 60mM-NaCl and ofbuffer containing 0.35 M-NaCl was applied, followed

175

L. G. Crossley and R. R. Porter

by a step to buffer containing 1.0M-NaCl. Thefractions containing C3bINA activity (Fig. 2abelow) were combined and made 60% saturated(36g/100ml) with (NH4)2SO4 and stirred for 2h at40C. After centrifugation of the mixture at 8000gfor 20min, the precipitate was dissolved in 60ml of50mM-sodium phosphate buffer, pH7.0, containing0.2M-NaCl, and then iPr2P-F was added to 2.5 mm.The concentrated solution was dialysed against three1-litre changes of buffer and passed through acolumn (4 cm x 7.5 cm) of wheat-germ agglutinin-Sepharose equilibrated with the same buffer at15 ml/h. After an initial wash with 1 column volumeof buffer, the flow rate was increased to 30ml/h, and25 ml fractions were collected until the A280 of theeluate was less than 0.15. Adsorbed protein waseluted with buffer containing 100mg of N-acetyl-D-glucosamine/ml (Fig. 2b below), or alternativelywith a linear gradient of 150ml of buffer and 150mlof buffer containing 30mg of N-acetyl-D-glucos-amine/ml followed by a step to buffer containing100mg of the sugar/ml (Fig. 2c below). The activefractions from stepwise elution were pooled, made2.5 mm with respect to iPr2P-F and dialysed againstthree 2-litre changes of 25 mM-potassium phosphatebuffer, pH 7.5. The solution was applied to a column(3.2cm x 15 cm) of hydroxyapatite equilibrated withthe dialysis buffer at a flow rate of 35 ml/h, and10ml fractions were collected. The column waswashed with buffer until the A280 of the eluate wasless than 0.05, when a linear gradient (2 x 300 ml) of25 mM-0.2 M-potassium phosphate buffer, pH 7.5,was applied (Fig. 2d below). The pooled fractionscontaining C3bINA activity were concentrated byprecipitation with (NH4)2SO4 (40g/100ml) asbefore, and the precipitate was redissolved in 3-5 mlof 25 mM-Tris/HCI buffer, pH 7.0. The concen-trated solution was passed directly through a columnof Sephacryl S-200 equilibrated with 25 mM-Tris/HCI buffer, pH7.0, containing 0.15M-NaCl at12 ml/h, and 4 ml fractions were collected (Fig. 2ebelow). The active fractions were combined, made2.5mM with respect to iPr2P-F and concentrated by

ultrafiltration with a Diaflo PM10 membrane to2-3 mg/ml and stored at 40C.

Electrophoresis in polyacrylamide gelsGel electrophoresis in 7.5% (w/v) polyacryl-

amide slabs in the presence of sodium dodecylsulphate was performed by the method of Laemmli(1970). Apparent molecular weights were assignedby comparison with proteins of known molecularweights (fIlH, mol.wt. 150000; transferrin, mol.wt.90000; bovine serum albumin, mol.wt. 67000;ovalbumin, mol.wt. 45000; bovine 16-lactoglobulin,mol.wt. 18400). Gel slabs were scanned with aVitatron TLD 100 densitometer equipped with apeak-area integrator.

Protein concentration and amino acid analysisThe recovery of protein during the purification of

C3bINA was determined by a modification of theLowry method (Hartree, 1972), with bovine serumalbumin as the reference standard. Other proteinconcentrations were calculated from the A280,assuming that an absorption value of 1.0 equals1 mg/ml or, for factor D, 0.5 mg/ml. Amino acidanalyses were carried out as described by Gigli et al.(1977).

Results

Haemolytic assay ofC3bINAThe optimal conditions for the inactivation of C3b

on EAC43 cells by C3bINA were investigated byusing pure C3bINA and,lH and veronal bufferswith a range of ionic strength and pH compatiblewith cell viability. EDTA was included in the veronalbuffers, as it is required to prevent C3b formationduring assays of crude fractions. At physiologicalpH, decreasing the ionic strength from 0.15M-NaClto 0.038M-NaCI resulted in a 3-fold increase in thespecific activity of C3bINA. In the presence of0.038M-NaCl, however, decreasing the pH from 7.5to 6.5 resulted in a 30-fold increase in the specificactivity of C3bINA (Table 1). Accordingly the

Table 1. Effect ofionic strength andpH on C3bINA haemolytic activityThe activity of pure C3bINA was determined by incubation of C3bINA with EAC43 cells and 1 ug of fIlH in thebuffers indicated at 370C for 30min, followed by the addition of factors B and D and guinea-pig serum as describedin the Materials and methods section. The activity (units/mg) obtained for C3bINA in each buffer is expressed asa percentage of the activity obtained in the standard assay buffer.

Composition of buffer (by vol.)

5% Dextrose/GV buffer/0.1 M-EDTA, pH 7.5 pH0:10:1 7.55:5:1 7.5

7.5:2.5:1 7.57.5:2.5:1 7.07.5:2.5:1 6.5

C3bINAactivity(%)

123

20100

1980

176

Human C3b inactivator

low-ionic-strength buffer at pH 6.5 was adopted asthe standard assay buffer.

The effect of varying the concentration of ff1H onC3bINA activity is shown in Fig. 1. In the absenceof ff1H there was no inhibition of lysis by C3bINAand similarly, in the absence of C3bINA, even highconcentrations of fIlH (4,ug/assay) did not result ininhibition of lysis. The decrease in the number ofC3b haemolytic sites was directly proportional tothe concentration offIH in the range 0.0625-1 ,ug offflH/assay for a given concentration of C3bINA.The amount of 8lH added to the standard assay wastherefore fixed at 0.1 ml of solution containing lOpgof ff1 H/ml, i.e. 1 ,ug/assay. Relatively more ff1H wasrequired to achieve near-maximal inactivation of cellbound C3b as the concentration of C3bINA wasdecreased. Thus, in the presence of 1,pg off,flH,near-maximal inactivation of cell bound C3b wasobserved only at the highest concentration ofC3bINA, when the molar ratio flH/C3bINA was20: 1. At the two lower concentrations of C3bINA,even though the molar ratios of fflH to C3bINAwere increased to 40: 1 and 80: 1, the inactivationwas not complete.

Purification ofC3bINAThe recovery of C3bINA activity was found to be

optimal when plasma depleted of plasminogen ratherthan serum was used as the starting material for thepurification scheme. Chromatography on the affinityadsorbent lysine-Sepharose gave quantitativerecovery of C3bINA activity and prevented activa-

0-ce

C-c

._l

.r_

IfliHi (ug/mI)Fig. 1. Effect of increasing concentration of 1H on

C3bINA inhibition ofhaemolytic activityEAC43 cells were incubated in EDTA/DGV buffer,pH 6.5, containing various concentrations of homo-geneous preparations of C3bINA [Oug (0);0.007,g (0); 0.O15,ug (O); 0.029,ug (E)l and /IH,and the haemolytic activity was determined as

described in the Materials and methods section.

Vol. 191

tion of the clotting factors. As an additionalprecaution against proteolysis, repeated addition ofiPr2P-F was made during the early stages of thepurification. Attempts to obtain an initial frac-tionation of the C3bINA activity in plasma by usingeither poly(ethylene glycol) or (NH4)2SO4 wereunsatisfactory, *since C3bINA activity was pre-cipitated over a wide range of poly(ethylene glycol)concentrations at pH7.4, affording little purifica-tion, and similarly at least 50% saturation with(NH4)2S04 was required to precipitate 80% of theC3bINA activity.

Chromatography of the plasminogen-depletedplasma on QAE-Sephadex A-50 removed 95% ofthe protein with little loss of C3bINA activity (Table2) and was therefore adopted as the initial purifica-tion step. Under the conditions specified, C3bINA,together with transferrin, which serves as a visualmarker, is separated from the bulk of the proteinbound to the resin by extended washing beforeapplication of the salt gradient (Fig. 2a). Analysis byimmunodiffusion showed that the QAE-SephadexA-50 pool of C3bINA activity did not contain fflH,which is eluted at higher salt concentration. Removalof fflH is advantageous before chromatography onwheat-germ agglutinin-Sepharose, as this protein ispresent in large amounts in plasma and was found tobind to the affinity resin as does C3bINA.

The QAE-Sephadex pool of C3bINA activity wasconcentrated by precipitation by 60% saturationwith (NH4)2SO4 before chromatography on wheat-germ agglutinin-Sepharose. Most of the transferrinstays in solution during the (NH4)2SO4 step, and theremainder is removed by chromatography on theaffinity adsorbent. The protein bound to wheat-germagglutinin-Sepharose was eluted by the inclusion ofN-acetyl-D-glucosamine in the buffer either as a stepto a sugar concentration of 100mg/ml (Fig. 2b) oras a gradient to a sugar concentration of 30mg/ml,which eluted the C3bINA activity as a diffuse peak,followed by a step to 100mg/ml to remove residualadsorbed protein (Fig. 2c). The simple step-elutionmethod gave a more concentrated protein solutionwith a better recovery of C3bINA activity than thegradient elution method, and was used routinely. Ineither case, two further steps were required toremove remaining contaminants: chromatographyon hydroxyapatite (Fig. 2d) was followed by gelfiltration on Sephacryl S-200 (Fig. 2e) as a final step.The solution of purified C3bINA was stable at 40Cover the pH range 6-8 for days and at pH 7 for aperiod of weeks, even in the absence of proteinaseinhibitors.

The purification scheme is summarized in Table 2,and electrophoresis patterns in polyacrylamide gelscontaining sodium dodecyl sulphate are shown inFig. 3. Electrophoresis of the C3bINA preparationobtained after fractionation on Sephacryl S-200

177

L. G. Crossley and R. R. Porter

Table 2. Purification ofC3bINAfrom plasmaThe purification was performed with 500 ml of plasma as described in the text.

FractionPlasmaQAE-Sephadex poolWheat-germ agglutinin-Sepharose poolHydroxyapatite poolSephacryl S-200 pool

Volume(ml)5007702239823

Totalactivity(units)44000038500015200011800088000

Totalprotein(mg)290001600150206

Specificactivity

(units/mg)15

24010005900

15000

Activityyield(%)10088352720

12 (a) N '1'

10

8 80 -

6 I/ 60U

4-' 40 .:I

2 -- -20 -

0 2000 4000 6000 8000

I(

(b)

0 400 800 1200

(c)/

1 400 800

120

_ 100 P

z 80 1,E __ 60,

20 .

._

-80

- 60 o

0-0

._:

-40

-20

revealed under non-reducing conditions a singleband of apparent mol.wt. 90000, and under reduc-ing conditions two bands of apparent mol.wts.52000 and 37000. This composition of two di-sulphide-linked polypeptide chains for C3bINA is inagreement with other reports (Fearon, 1977; Pang-burn et al., 1977). The C3bINA preparation wasestimated by densitometric scanning of the gels to be90-95% homogeneous, with a very faint impuritydue to incomplete separation on the Sephacryl S-200column. Under non-reducing conditions the impurityis a single band of apparent mol.wt. 160000, andunder reducing conditions two bands of mol.wts.60000 and 22000. The impurity, which may be IgG,could be removed by re-running on the Sephacrylcolumn. The yield of C3bINA was 6mg from 500mlof plasma (determined by the Lowry protein assaymethod with serum albumin as standard), with anoverall recovery of haemolytic activity of 20%.

0.6

040, 0.4

0.2

0.

0.

0.

120 160 200 240 280 320Volume of eluent (ml)

30

O

0

1-1 P

E - .gE

iioo I.- 80

.604go

X0 40,.

0 .e

0

400

tO.00 _

Fig. 2. Chromatography ofC3bINA(a) The dialysed preparation of plasminogen-depleted plasma (750ml) was loaded on a column(7cm x 36cm) of QAE-Sephadex and eluted withbuffer containing 20mM-Tris/HCl, pH7.8, and anincreasing concentration of NaCl applied as agradient (first arrow) and 1.OM-NaCl step (secondarrow) as described in the Materials and methodssection. Active fractions were pooled as shown.

, A280; 0, C3bINA activity (percentage inhibi-

tion of lysis); ----, conductivity (mS). (b) The poolfrom (a) was concentrated and dialysed (80 ml), thenapplied to a column (4 cm x 7.5 cm) of wheat-germagglutinin-Sepharose and eluted with 0.2M-NaCl/50mM-sodium phosphate buffer, pH 7.0, as des-cribed in the Materials and methods section. Thearrow indicates the addition of 100mg of N-acetyl-D-glucosamine/ml to the eluting buffer. Sym-bols are as for (a). (c) A pool obtained in a separatepreparation, but by the same procedure as that in(a), was loaded on wheat-germ agglutinin-Sepharose as in (b). Elution of adsorbed protein waseffected by increasing concentration of N-acetyl-D-glucosamine in buffer applied as a gradient (firstarrow) followed by a step to 100mg/ml (secondarrow). Symbols are as for (a). (d) After dialysis, thepool from (b) was loaded on a hydroxyapatitecolumn (3.2cm x 15cm) in 25 mM-potassium phos-phate buffer, pH 7.5, and eluted by a gradient ofincreasing phosphate concentration as described inthe Materials and methods section. Symbols are asfor (a). (e) The concentrated pool from (d) waschromatographed on a Sephacryl S-200 column(2.5cm x lOOcm) in 0.15 M-NaCl/25 mM-Tris/HClbuffer, pH 7.0. Symbols are as for (a).

1980

2.5

2.0

1.5

1.0

0.5

, (d) 3

;, a_ V3

I-I0 200 400 600 800 1000 1200

.6 (e)

2-~~~~~~~~~~~~~~~~

178

000C-4

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000C4

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a

x0s

1.CO

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11

v

Human C3b inactivator

W~~~

... ...

(1) (2) (3) (4) (5) (6) (7)

Fig. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate of samples taken at various stagesofthe C3bINA purification

For experimental details see the text. Reduced samples: (1) plasma; (2) QAE-Sephadex pool; (3) wheat-germagglutinin pool; (4) hydroxyapatite pool; (5) Sephacryl S-200 pool (4,ug). Non-reduced samples: (6) Sephacryl S-200pool (2pg); (7) Sephacryl S-200 pool (10,g).

Cleavage of fluid-phase C3b and inactive C3 byC3bINA and JJHAs shown in Fig. 4, the a'-chain of C3b in

solution was cleaved by C3bINA and J1IH toproduce two fragments (which required reductionfor dissociation from the fl-chain) of apparentmol.wts. 67000 and 43000. The a'-chain of C3bwas unaffected by either C3bINA or,ilH alone. Noother degradative products have been observed evenin the absence of iPr2P-F treatment of the purifiedcomponents. With equimolar concentrations of C3b,C3bINA and /J1H at pH7, 50% cleavage of C3ba'-chain occurred after about 1 min at 370C (Fig.5). When the molar concentration of either C3bINAor JlH was decreased 10-fold, the time required for50% cleavage was increased approx. 10-fold andmore than 30-fold respectively.

Native C3, unlike C3b, is not cleaved by C3bINAand fllH. However, preparations of C3 lose haemo-lytic activity on standing at 40C or on slow-freezingat -200C (von Zabern et al., 1980; M. A. Kerr,unpublished work). The loss of haemolytic activitycorrelates with the loss of hydrolysis of the a-chainby C3 convertases of both activation pathways (C.Parkes, R. G. DiScipio & M. A. Kerr, unpublishedwork). This denaturation of native C3 is accom-panied by exposure of a reactive thiol group, asoccurs when C3b is formed (Janatova et al., 1979).Such inactive C3 was also found to be cleaved by

Vol. 191

/31H !v

( _ _

C3blNA

(1) (2) (3) (4) (5) (6) (7) (8) (9)(10)(1 1((12)

Fig. 4. Cleavage offluid-phase C3b by C3bINA and alHSodium dodecyl sulphate / polyacrylamide - gelelectrophoresis of reduced samples was performedas described in the text: (1) C3b (1.3,ug); (2)C3bINA (4,ug); (3) PlH (l.5,ug). Other samples(20,ul) in 0.15 M-NaCI/O. I M-sodium phosphatebuffer, pH 7, containing equimolar concentrations ofC3b (1.3,ug), C3bINA (0.6,ug) or lIH (0.9,ug)added as indicated, were incubated at 37°C forvarious times, then reduced and electrophoresed: (4)C3b+C3bINA, 30min; (5) C3b±+llH, 30min;(6)-(12) C3b, C3bINA +#lH [(6) 2min; (7) 4min;(8) 6min; (9) 10min; (10) 15min; (I ) 20min; (12)30minl. Arrows indicate the C3bINA cleavageproducts of apparent mol.wts. 67 000 and 43 000.

179

L. G. Crossley and R. R. Porter

Ca60

u

C4.-000

Cu

20

0 10 20 30Time (min)

Fig. 5. Rate ofcleavage of C3b as afunction ofC3bINAand JJJH concentration

Samples in 0.15 M-NaCI/O. 1 M-sodium phosphatebuffer, pH7, containing C3b, C3bINA and J1H invarious molar proportions [1: 1: 1 (0); 1 :0.1: 1(0E; 1:1 :0.1 (A)1 were incubated for various timesat 37°C, then electrophoresed. The percentagecleavage of C3b a '-chain was determined bydensitometric scanning of the slab gels.

/)1H - -

a _ _ w_v w- -

.4e k__ _" _40

C3blNA ee... V : 40qw.

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)(11)(12)

Fig. 6. Cleavage offluid-phase inactive C3 by C3bINAand fJlH

Sodium dodecyl sulphate / polyacrylamide - gelelectrophoresis of reduced samples was performedas described for Fig. 4 except that inactive C3instead of C3b was examined. Arrows indicate theC3bINA cleavage products of apparent mol.wts.75 000 and 43 000.

C3bINA and f,1H to yield the pattern shown in Fig.6. As was observed with C3b, the a-chain ofinactive C3 was unaffected by either C3bINA orfflH alone. Two fragments were produced bycleavage of the a-chain, and these had apparentmol.wts. of 75 000 and 43 000. Since the C3a regionof C3 is at the N-terminus of the a-chain (Hugli,1975; Tack et al, 1979), and since cleavage of C3byields fragments of the a'-chain of mol.wts. 67000and 43000, this result indicates that the 43000-mol.wt. fragment is the C-terminal fragment of thea- and a'-chains. Comparison of the rates ofcleavage of the a'-chain of C3b and of the a-chainof inactive C3, at two different concentrations ofC3bINA, indicated that inactive C3 was cleaved atabout one-quarter of the rate of C3b (Fig. 7).

pH optimumfor C3bINA activityThe pH optimum for the cleavage of C3b by

C3bINA and f,lH was found to be 6 when examinedin various buffers in the pH range of 5-9 (Fig. 8).

.-

-00-S

CI

.0ceco

-0clC._

00(Uococc.9

0 10 20 30Time (min)

Fig. 7. Comparison of the rate ofcleavage of inactive C3and C3b

Samples in 0.15 M-NaCl/0. 1 M-sodium phosphatebuffer, pH 7, containing inactive C3, C3bINA andJIH [in molar proportions 1:1:1 (0) and 1:0.1:1(01 or C3b, C3bINA and /1H [in molar pro-portions 1:1:1 (40) and 1:0.1:1 (E)] were incu-bated for various times at 37°C, then electro-phoresed. The percentage cleavage of inactive C3a -chain or C3b a'-chain was determined bydensitometric scanning of the gel slab.

1980

180

Human C3b inactivator

-

-

C%

0

>I )

co -30 E

201- 20I ~~~~~~~~~~105 6 7 o_

pH

Fig. 8. Effect ofpH on the cleavage offluid-phase C3Bby C3bINA and fJlH

Samples containing C3b, C3bINA and ,IlH (inmolar proportions 1 :0.02:1) were incubated for7.5min at 370C in buffers [A, 0.1 M-Mes (4-morpholine-ethanesulphonic acid); B, 0.1 M-Pipes(1,4-piperazinediethanesulphonic acid); C, 0.1 M-Trisl adjusted to the indicated pH and conductivity(---- ) with NaOH or HCI. After electrophoresis,the percentage cleavage of the C3b a'-chain wasdetermined by densitometric scanning of the gelslabs.

This agrees with the optimum pH of 6.5 (in thelimited range 6.5-7.5) that gave the maximalinactivation of C3b on EAC43 cells (Table 1).

Effect ofproteinase inhibitors on C3bINAPreliminary attempts to identify the class of

proteinase to which C3bINA belongs were un-successful. As judged by splitting of the C3ba'-chain, C3bINA was not inhibited by iPr2P-F(10 mM), 7-amino- 1-chloro-3-tosylamidoheptan-2-one (5 mM), phenylmethanesulphonyl fluoride(0.1 mM) or benzamidine (5 mM), and hence may notbe a serine esterase-type proteinase. However, C2and factor B, which have been reported to be serineproteinases, are also resistant to some of thesereagents (Kerr, 1979). Chelating reagents such as1,10-phenanthroline (1 mM), EDTA (50mM) andEGTA (50mM) had no effect, and further evidenceagainst a functional role for metals was observed inthe acidic pH optimum of 6 for the reaction. Therewas no evidence for an essential thiol group, as therewas no inhibition by HgCl2 (1 mM), iodoacetic acid(1 mM) or 4-chloromercuribenzoate (0.1 mM), al-though N-ethylmaleimide showed some inhibition ata concentration of 1 mm.

Strong inhibition of C3bINA was observed with1 mM-dithiothreitol and 10mM-2-mercaptoethanol,presumably as a result of the reduction of interchain

disulphide bonds. Surprisingly, ZnCl2, but notMgCl2, MnCl2, CaCl2, CoCl2 or NiCl2, all at 1 mM,strongly inhibited C3bINA. The mechanism of thiseffect of ZnC12 has not been investigated.The C3bINA proteolytic activity is unusual in

that it appears to be dependent on prior interactionof either ,B1H or C4-binding protein with thesubstrates C3b or C4b and in the specificity of thehydrolysis of a single peptide bond in C3b a'-chainand oftwo bonds in the C4b a'-chain.

Discussion

Inactivation ofC3bThe assay of C3bINA activity measures the

inactivation of C3b bound to EAC43 cells. Onaddition of factors B and D, in the presence of Mg2+and Ca2+, the alternative-pathway C3 convertase isformed, and the subsequent addition of C3-C9 asguinea-pig serum diluted in EDTA results inactivation of the terminal components causing celllysis. In the presence of limiting amounts of C3b onthe EAC43 cells and an excess of factors B, D andguinea-pig serum, cell lysis is inhibited to a degreedetermined by the extent of inactivation of C3b byC3bINA. When pure C3bINA was assayed withpure Ji1H, the inactivation of cell bound C3b wasenhanced 100-fold in a low-ionic-strength pH6.5buffer as compared with an iso-osmotic pH 7.5buffer. This result is in agreement with similarstudies with serum as a source of C3bINA and f1H(Whaley et al., 1976).The inactivation of C3b in both solid and fluid

phases does not proceed in the absence of JJlH. Anabsolute requirement for fJ1H by C3bINA forcleavage of C3b in solution was first reported byPangburn et al. (1977). In other studies, however,both C3bINA alone and/or,IlH alone have beenreported to produce concentration-dependent inhibi-tion of solid-phase C3b, with 1IH serving toaccelerate the effect of C3bINA (Whaley & Ruddy,1976; Fearon & Austen, 1977; Fujita &Nussenzweig, 1979).

Evidence suggesting that C3bINA binds pre-ferentially or exclusively to a bimolecular complex ofC3b and JJ1H, as compared with C3b alone, wasobtained in experiments with both solid-phase andfluid-phase C3b. Thus relatively more 161H wasrequired to achieve maximum inactivation of cellbound C3b as the concentration of C3bINA wasdecreased. Similarly, the cleavage of C3b in solutionwas slowed more by a decrease in fIlH con-centration than by a corresponding decrease inC3bINA concentration. The conclusion that the rateof inactivation of C3b by C3bINA is a function ofthe concentration of the C3b-fIH complex issupported by the results reported for binding studieswith the solid-phase EC3b. At 0°C, C3bINA

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182 L. G. Crossley and R. R. Porter

binding was proportional to the amount of,1BHbound and approached equimolar amounts atsaturation of the sites binding C3bINA, with up to30-fold enhancement of C3bINA binding in thepresence of f1H (Pangburn & Miiller-Eberhard,1978).

Purification ofC3bINALoss of C3bINA activity during the isolation was

little more than handling losses, except in the bindingand desorption from the wheat-germ agglutinin-Sepharose column, where it was about 50% evenwith stepwise elution (Table 2). This may have beendue to increased susceptibility of C3bINA toproteolytic digestion when bound even thoughinhibitors were added, but perhaps is more probablydue to irreversible adsorption. Attempts to improvethis yield are continuing, but the overall 20%recovery, described here, is higher than in pre-viously described methods (Pangburn et al., 1977;Fearon, 1977).

Hydrolysis ofinactive C3 by C3bINA and/#HThe hydrolysis of the a '-chain of C3b by

C3bINA and ,61H and of C4b by C3bINA andC4-binding protein has been described previously(Pangburn et al., 1977; Nagasawa & Stroud, 1977;Fujita et al., 1978), and has been confirmed here.Native C3 and C4 are not hydrolysed by theseenzymes, but both proteins lose haemolytic activityon standing in the cold, on freezing and thawing oron treatment with amines. In the last-mentionedcase, the inactive protein is antigenically similar toC3b or C4b, though no peptide bond is split (vonZabern et al., 1980). We have found that inactive C3is hydrolysed by C3bINA and M1H, though moreslowly than C3b, to give chain products of 75000and 43000mol.wt., rather than 67000 and43000 mol.wt. as from the a'-chain of C3b (Fig. 7).As discussed above, this suggests that the 43000-mol.wt. fragment is C-terminal and that the largerfragment is N-terminal.

Similarly, inactive C4 but not native C4 ishydrolysed by C3bINA and C4-binding protein(E. M. Press, unpublished work), and this suggeststhat the denaturation ofC3 and C4 results in a changeof conformation similar to that of C3b and C4b. Thisis in agreement with the evidence obtained by vonZabern et al. (1980) and with the observation byJanatova et al. (1979) that a thiol group unreactivein native C3 or C4 becomes reactive in both C3bor C4b and in inactive C3 and C4.

The gifts of anti-(human fl1H) serum (sheep) by Dr. A.R. Bradwell of the Immunodiagnostic Research Labora-tory, Department of Immunology, University of Birming-ham, Birmingham, U.K., and of complement compon-

ents by Dr. D. L. Christie, Dr. R. G. Di Scipio, Dr. M. A.Kerr and Dr. R. B. Sim of this laboratory are gratefullyacknowledged.

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1980