Purification and Some Properties of Factor {ieD} of the Human Properdin System

Z. Immun.-Forsch. vol. 152, pp. 231-243 (1976) Max-Planck-Institut fUr experimentelle Medizin, Department of Biochemical Pharmacology, Gottingen, Federal Republic of Germany

Purification and Some Properties of Factor D of the Human Properdin System LOTHAR DIEMINGER, WALTER VOGT, and RAINER LYNEN With 9 Figures Received May 26, 1976 . Accepted in Revised Form August 9, 1976

Abstract Factor f> has been purified by gel and ion exchange chromatographies, and by ultrafiltration through different membranes. The final preparation appeared pure in various analytical tests. The molecular weight of human f> is 21,500 according to gel chromatography, the isoelectric point was found at pH 7.S. Factor f> is an active esterolytic enzyme, it cleaves N-IX-acetyl-L-Iysine methyl ester and N-IX-acetyl-L-glycyl-L-Iysine methyl ester. Both peptide esters inhibit the hydrolytic activation of factor B by f> in the presence of cobra venom factor. f> is also inhibited by diisopropyl-fluorophosphate and by phenylmethyl-sulfonyl-fluoride.

Introduction The properdin system represents one of two pathways by which the terminal complement sequence is activated, and biologically active humoral factors are released. A highly efficient C 3 activating system is generated when factor B, C3b and factor D interact (1). Factor D has been recognized as an active, DFP-inhibitable enzyme (2) and the mechanism of interaction of the three components has been elucidated: D cleaves B when this is bound to C3b, and the resulting complex C3b, Bb (C3B) is capable of cleaving (activating) C3 (3). Although human factor D has been purified and used in this laboratory for more than three years, a detailed description of the purification and of general properties had not yet been given. They are presented in this communication. Further some studies on enzymatic properties of factor D are included. Abbreviations used: CVF = cobra venom factor, ALME = N -IX-acetyl-L-Iysine methyl ester HCI, AGLME = N -IX-acetyl-L-glycyl-L-Iysine methyl ester acetate, DFP = diisopropyl-fluorophosphate, PMSF = phenyl-methyl-sulfonyl-fluoride.

232 . L. DIEMINGER, W. VOGT, and R. LYNEN

After this work was completed an abstract describing the purification of human factor D by affinity chromatography was published (4).

Materials and Methods Human serum Human serum was pooled after centrifugation of spontaneously clotted blood and stored at -70°C.

Cobra venom factor (CVF) CVF was purified from Naja naja venom by ion exchange and gel chromatography (5). Batches were stored at -70°C in 20 mM Tris/HCI pH 7.4.

Factor B This protein was isolated from human serum and purified as described earlier (6). It appeared pure in alkaline disk electrophoresis (100 ftg applied) and immunoelectrophoresis against anti-whole human serum. Batches were stored at -70°C in barbital buffered saline pH 7.4 containing 5% glucose.

Factor Jj Batches of 500 ml human serum containing 5 mM EDTA were passed througr. a column (20 X 60 em) of Sephadex G-200 equilibrated with 0.15 M NaCI solution containing 4 mM NaN 3 • .i5 activity was recovered in fractions between the albumin and a later eluting small peptide peak. These fractions were pooled, passed through an Amicon filter XM50 and the filtrate was then concentrated by ultrafiltration through an Amicon filter UM 2. The concentrated retentate was dialysed against 5 mM potassium phosphate buffer pH 6.0, adjusted to a conductivity of 5 mS (O°C) by addition of NaCI and applied to a column (1.3 X 5 em) of SP-Sephadex G-25 equilibrated with the same buffer. Factor b activity eluted in a linear salt gradient to 5 mM potassium phosphate buffer pH 6.0 containing 0.5 M NaCl at a conductivity of 10 mS (0° C). The active fractions were stored at -70° C in the eluting buffer.

Complement components For the immune haemolytic estimation of C 3 functionally pure complement components from guinea pig serum (Cordis Corporation, Miami, Fla.) were used. The components were dissolved as suggested by the manufacturer, so that undiluted solutions of C 1 contained 10,000 haemolytic units/ml, C 2-C 9 1000 units/ml.

Barbital buffered saline When used as diluent and medium in immune haemolysis experiments this solution contained 0.076 M NaCI, 2.5 mM barbital buffer pH 7.4, 1 mM Mg++, 0.15 mM Ca++, 0.139 M glucose and 0.1 % gelatin (7). When used for other purposes glucose and gelatin were omitted.

Synthetic substrates for factor Jj N -ex-acetyl-L-Iysine methyl ester HCI (ALME) was obtained from Sigma Chemical Company, St. Louis, Mo., and N-ex-acetyl-L-glycyl-L-lysine methyl ester acetate (AGLME) from Protein Research Foundation, Osaka, Japan. Diisopropyl-jluorophosphate (DF P) and phenyl- methyl- sulfonyl-jluoride (PMSF) were obtained from Serva, Heidelberg, Germany.

Purification and Some Properties of Factor

D.

233

Functional assay for factor 15 activity D was assayed by its capacity to generate the C 3 cleaving enzyme CVFB from CVF and B in the presence of Mg++ (8, 9). In a certain range, the amount of CVFB complex formed depends on the amount of factor D present and the former can be assayed by its C 3 cleaving capacity. Varying amounts of D were incubated with 2 fig Band 3 fig CVF in the presence of 2.5 mM Mg++ in a total volume of 95 fll made up with barbital-buffered saline without glucose and gelatin, for 30 min at 37°C. After stopping further complex formation by addition of 5 fll 0.3 M EDTA (final concentration 15 mM) 25 fll Cordis C3 g. p. were added and incubated for 30 min at 30° C. C 3 cleavage (loss of C 3 activity) was then assayed by immune haemolysis as described earlier (9). Figure 1 shows a dose-response curve of D obtained in this way. Standardization of 15 activity One unit of D was defined as the amount of factor D which causes complex formation from 4 fig Band 6 fig CVF in an amount that cleaves 50% of 25 Cordis units C 3 during incubation for 30 min at 37 °C. Pretreatment of factor 15 with inhibitors Stock solutions of DFP (5 X 10-3 M) and PM SF (2 X 10-3 M) were prepared in 10% isopropanol and kept at -20°C. 1 volume of the stock solution was added to 9 vol factor D in buffer pH 6.0 and the mixture was kept at 30°C for 30 min. At the concentrations used (1 % during inhibitor treatment, 0.1 % during incubation of treated D with substrates) isopropanol enhanced the B cleaving activity of D considerably; therefore control incubations with non-inhibited D were made by pretreating the control D preparation with inhibitor-free isopropanol solution in the same way. Antigen-antibody-crossed immunoelectrophoresis This method was used for quantification of factor B, as described earlier (10). For assay of B in mixtures where CVFB complexes had formed sufficient

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Fig. 1. Dose-response curve of factor D. Constant amounts of Band CVF were incubated with increasing amounts of D (abscissa). Then EDTA and C3 was added and C 3 cleaving activity (ordinate, loss of C 3) of the CVFB complex formed was assessed by haemolytic titration of residual C 3 (see methods).

234 . L. DIEMI:NGn.R, W. VOGT, and R. LYNEN rabbit anti·CVF serum was added to the sample to bind and dissociate any CVFB complexes which could otherwise interfere with the estimation of in· tact B. The Bb fragment once released from CVF runs to the cathode and thus does not interfere with B estimation.

Zone electrophoresis Micro zone electrophoresis was performed on strips of cellulose acetate (0.15 mm thick) in 90 mM barbital buffer pH 8.6. Protein bands were stained with 0.2% Ponceau S in 3% trichloroacetic acid and 3% sulfosalicylic acid. Disc electrophoresis Acid disc electrophoresis was performed in gels containing 7.5% polyacrylamide pH 4.3 (gel system number 7) (11). Gels were casted on a normal scale (5 mm diameter) or in micro caps (0.5 mm diameter), the latter with a linear density gradient rising from 2-25% polyacrylamide (12). The micro disc electrophoresis was kindly performed by Dr. W. DAMES, Dept. of Neurophysiology in this Institute. Thin layer chromatography For demonstration of the cleavage products of synthetic substrates we used thin layer chromatography on prefabricated plates (TLC aluminium sheets silica gel 60, Merck, Darmstadt, Germany). The solvent mixture was methylethyl-ketone/pyridine/acetic acid/water: 70/15/2/15 (volume ratios). The peptide esters and their products were stained with ninhydrine. I soelectric focusing For determination of the isoelectric point purified factor D was subjected to isoelectric focusing in 2% ampholine pH 6-9, using a gradient of glycerol (0 to 60%) for stabilization. The position of D in the fractions obtained was assessed by functional assay.

Results Properties of factor D A summary of D purification is shown in Table 1. The elution profile obtained by the last purification step, chromatography on SPSephadex, is seen in Figure 2. Preparations obtained were checked for Tab. 1. Purification of factor

D.

Stage

Vol. (ml)

Protein (mg)

D Activity (Units)

Yield

Specific Activity (Units/mg)

Human Serum *) Aftcr G200 Filtrate XM 50 Retentate UM2 After SP-Seph.

500 2390 2370 90 7.5

52,500 8430 120 90 0.25

1,195,000 282,750 227,200 86,250

100% 26% 20% 7.6%

142 2388 2490 345,000

*) It was not possible to quantify factor D in whole serum by the described method. When a semipurified sample of D was passed through Sephadex under the same conditions as whole serum a loss of 50% D activity occurred. Provided this loss would be the same as in whole serum all yields would be halved when related to whole serum.

Purification and Some Properties of Factor D . 235

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Fig. 2. Chromatography on SP-Sephadex of factor D, last step of purification. Protein distribution shown as O.D' 280 (0--0). D activity estimated as loss ofC3 (see Methods) (e--e). Conductivity (mS at O°C) (---).

purity on polyacrylamide gels in disc electrophoresis. Alkaline gels did not show any band, in acid gels one single band appeared. After micro disc electrophoresis of D in continuous gel concentration gradients, again a single band appeared (Fig. 3). Attempts to elute D activity from polyacrylamide gels were unsuccessful. Therefore, samples of purified D were run on cellulose acetate strips in micro zone electrophoresis. Again a single band was detected after staining. Unstained

'~'>;;""'" Fig. 3 . Upper gel: Acid disc electrophoresis of purified factor D. Lower gel; Acid microdisc electrophoresis on continuous polyacrylamide concentration gradient ranging from 2 to 25%. In both gels start and anode at the right.

236 .

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and R.

LYNEN

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Fig. 4. Electrophoresis of purified factor D on a strip of cellulose acetate. Top band: 2.5 ftg D; middle band: 10 ftg human serum albumin; lower band: 0.25 ftl human serum. Superimposed diagram: D activity (given as loss of C 3) in fractions eluted without staining from a pars.llel strip run with D. Start at arrow.

strips were eluted, the eluates of the band region exhibited factor D activity (Fig. 4). Human factor D has a molecular weight of 21,500 ± 700 S.D. as estimated by gel filtration on columns of Sephadex G 100 and G 75 (1.5 X 90 cm) calibrated with ovalbumin (45,000), cytochrome C

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Fig. 5. Immunoelectrophoresis of purified factor f>; lower well: 20 ftl human serum; through: rabbit anti·D y.globulin. Anode to the right. The faint arc below the trough is human y.globulin to which the antiserum contained some antibody.

Purification and Some Properties of Factor f> . 237

(12,000) and chymotrypsin (25,000). The isoelectric point was found at pH 7.8 by isoelectric focusing. In immunoelectrophoresis factor D has ~- to y-mobility (Fig. 5).

Interaction of peptide esters with factor D Inhibition of D activity on factor B by ALME and AGLME Both ALME and AGLME inhibit the generation of C 3 cleaving activity by D. In the experiment shown in Figure 6 20 flg B, 30 flg CVF and 5.5 flg D were incubated at 37°C in a total volume of 800 fll containing 2.5 mM Mg++, with or without 2.6 mM ALME or 2 mM AGLME, respectively. At times indicated in the figure samples of 95 fll were withdrawn and added to 5 fll 0.3 M EDTA, to stop further complex formation. Then 25 fll C 3 was added to each sample, and C 3 cleavage during further incubation for 30 min at 37°C was determined. ALME did not inhibit the activity of preformed CVFB on C 3 (Tab. 2), indicating that it interferes with the activation of B by D. The inhibition is concentration-dependent. Under the conditions chosen 50 % inhibition of D activity was achieved by 26 mM ALME and 20 mM AGLME. Inhibition of B cleavage by D was shown directly, by measuring residual B in mixtures containing 4.5 flg B, 0.2 flg V, 9 flg CVF, 5 mM Mg++ in a total volume of 15 fll with or without 17 mM ALME or 13 mM AGLME. The mixtures were incubated for 30 min at 37°C,

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[min]

Fig. 6. Inhibition of f> avtivity by ALME ( . - - . ) , respectively. Control

(A-A)·

238 . L. DIEMINGER, W. VOGT, and R. LYNEN Tab. 2. Effect of ALME on C 3 cleaving activity of preformed CVFE complex. No.

Complex

Additions

1

CVFE CVFE CVFE (ALME) CFV

EDTA EDTA EDTA EDTA

2 3 4

+

Consumption of C3 (%) 80 79 37 3

ALME

CVFE complexes were prepared by incubation of CVF, B and :is as described in methods. Then EDTA, 26 mM ALME where indicated, and C 3 were added and C3 cleavage during 30 min at 37°C was estimated. For comparison, the activity of one sample of CVFE which had been generated in the presence of ALME is shown (No.3), and one sample lacking B (No.4).

and aliquots of 8 ,ul used for rocket electrophoresis, after addition of anti-CVF serum (see Methods). In a typical experiment of this kind B cleavage amounted to 50% in the absence of peptide esters, while

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Fig. 7. Cleavage of ALME by :is or trypsin, respectively. Thin-layer chromatograms of reaction mixtures. Upper chromatogram: 1. ALME control; 2. ALME + :is; 3. ALME + :is pretreated with DFP; 4. ALME + :is pretreated with PMSF; 5. N -cx-acetyl-L-lysine; 6. L-lysine. Lower chromatogram: Same Incubation mixtures as in upper chromatogram but :is replaced by trypsin.

Purification and Some Properties of Factor f> . 239

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Fig. 8. Cleavage of AGLME by f> or trypsin, respectively. Thin-layer chromatograms of reaction mixtures. Upper chromatogram: 1. AGLME control; 2. AGLME + f>; 3. AGLME + f> pretreated with DFP; 4. AGLME + f> pretreated with PMSF; 5. N -ex-acetyl-L-glycyl-L-lysine; 6. L-glycyl-L-lysine. Lower chromatogram: Same incubation mixtures as in upper chromatogram but f> replaced by trypsin.

only 20% were cleaved in the presence of ALME and 15% in the presence of AGLME.

Oleavage of peptide esters by factor D 50 p,g ALME or AGLME were incubated with 0.5 p,g factor D in a total volume of 17 p,l 10 mM phosphate buffer pH 7.0, for 30 min at 30°C. Analysis of the reaction mixture by thinlayer chromatography showed that the mobility of the esters was increased by the treatment indicating an enzymic attack. Pretreatment of D with one of the inhibitors DFP or PMSF prevented the effect (Fig. 7 and 8). Trypsin which cleaves the ester linkage of the used substrates was investigated for comparison; as expected the products of trypsin treatment had the same mobility as authentic N -acetyl-lysine and N -acetyl-glycyl-Iysine, respectively, i.e. a far lower RF value than the corresponding esters (Fig. 7 and 8). The higher mobility of the reaction products obtained after treatment of the esters with D indicates a less polar compound.

240 . L. DIEMINGER, W. VOGT, and R. LYNEN

NMR spectra of the products, kindly made by Mr. B. SEEGER, Dept. of Chemistry of this Institute, proved that the ethyl ester was cleaved by D; the singlet characteristic of the methyl ester group had disappeared in the spectra of the reaction products. In agreement with the ester cleavage free methanol was detected in the reaction mixture of ALME and D by gas chromatographic analysis (kindly performed by Dr. J. DITT, Gerichtsmedizinisches Institut, University of G6ttingen). The fact that subsequent treatment of the unknown product with trypsin resulted in a compound indistinguishable from the direct trypsin product suggested that after cleavage of the ester by D the liberated carboxyl group of lysine formed a new linkage - trypsinsensitive but D-insensitive - with some other group. However, NMR spectra gave no indication about its nature. Inhibition of D activity by DFP and PMSF

Pretreatment of factor D with DFP (5 X 10-4 M) or PMSF (2 X 10-4 M) led to a considerable reduction of its potency to generate C 3 cleaving activity on incubation with CVF and B (Fig. 9). A total block

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Fig. 9. Effect of PMSF and DFP on f> activity, assayed by estimating the potency of f> to generate a C 3 cleaving CVFB complex. 5.5 pg f> pretreated with one of the inhibitors or with isopropanol dilution alone (control) were incubated with 20 pg Band 30 pg CVF in a total volume of 800 pI containing 2.5 mM Mg++, at 37°C. At the indicated times 95 pI aliquots were removed, further complex generation was stopped by addition of 5 pI 0.3 M EDTA, and 25 pI C 3 were added to each sample which was subsequently incubated for 30 min at 37°C.

Purification and Some Properties of Factor f> . 241 Tab. 3. Effect of DFP- or PMSF-pretreatment of f> on its capacity to cleave B in presence of CVF.

No.

Pretreatment of f>

Cleavage ofB (%)

1

1 % isopropanol 5 X 10-4 M DFP-isopropanol 2 X 10-4 M PMSF -isopropanol

78 30

2 3

32

The incubation mixtures contained 0.2 /1g f> pretreated as indicated, 4.5/1g B, 9/1g CVF and 5 mM Mg++ in a total volume of 15/11. Cleavage of B was estimated by rocket electrophoresis.

was, however, never observed. The activity of preformed CVFB complexes on C 3 and the assay of residual C 3 activity were not affected by the presence of relevant amounts of DFP, PMSF, or isopropanol which was introduced with the inhibitor solutions. The effect of the inhibitors is thus due to inhibition of D. This is also evident from the direct assay of B cleavage by D in the presence of CVF (Tab. 3). As shown in Figures 7 and 8 PMSF and DFP inhibited also the peptide ester-cleaving activity of D. Discussion Factor D as purified from human serum is an active hydrolytic enzyme. FEARON et al. (2) discovered that its potency to activate B is inhibited by DFP, indicating a serine esterase. Further its activating effect on B was found to be analogous to and replaceable by trypsin (9) and other hydrolytic enzymes (13). However, a substrate that would be directly attacked by D was hitherto unknown. Factor B is cleaved by D only in the presence of C3b or CVF (9, 3). The present studies now demonstrate directly the enzymic nature of D; without any co-factors it attacks ALME and AGLME. This activity, like that on B, is inhibited not only by DFP, but also by PMSF. While the nature of the products obtained from interaction of D and the peptide esters is still unknown, it is clear that the effect of D involves cleavage of the methyl ester bond, i.e. D has esterolytic potency. The inhibitory effect of the 2 peptide esters used on the generation by D of a B-dependent C 3 convertase is likely to be due to competition between B and the ester. However, this mode of inhibition has not yet been proved. In its physicochemical properties human factor D differs to some extent from guinea pig D. While guinea pig D has its isoelectric point between pH 9.35 (14) and pH 9.5 (15) the isoelectric point for human

242 . L. DIEMINGER, W. VOGT, and R. LYNEN

D was found at pH 7.8. On the other hand the molecular weight of both proteins is of the same order and there are even immunochemical similarities (10). Acknowledgements We thank Mrs. J UTTA GLOTH, Miss ELISABETH GRUNEFELD and Mrs. HEIDRUN PRZYKLENK for their excellent technical assistance.

References I. MULLER-EBERHARD, H. J., and O. GOTZE. 1972. C3 Pro activator convertase and its mode of action. J. expo Med. 135: 1003. 2. FEARON, D. T., K. F. AUSTEN, and S. RUDDY. 1974. Properdin factor D: Characterization of its active site and isolation of the precursor form. J. expo Med. 139: 355. 3. VOGT, W., G. SCHMIDT, L. DIEMINGER, and R. LYNEN. 1975. Formation and composition of the C 3 activating enzyme complex of the properdin system. Sequential assembly of its components on solid-phase trypsin-agarose. Z. Immun.-Forsch. 149: 440. 4. GOTZE, O. 1976. Purification of factor D of the properdin (P) system by immune adsorbent chromatography. Fed. Proc. 35: 254. 5. BALLOW, M., and C. G. COCHRANE. 1969. Two anticomplementary factors in cobra venom: Hemolysis of guinea pig erythrocytes by one of them. J. Immunol. 103: 944. 6. LYNEN, R., V. BRADE, A. WOLF, and W. VOGT. 1973. Purification and some properties of a heat-labile serum factor (UF): Indentity with glycine-rich ~-glycoprotein and properdin factor B. Hoppe-Seyler's Z. Physiol. Chern. 354: 37. 7. MAYER, M. M. 1961. Complement and complement fixation. In: Experimental Immunochemistry (Ed. by E. A. KABAT, and M. M. MAYER), p. 133. Thomas, Springfield, Ill., 1961. 8. HUNSICKER, L. G., S. RUDDY, and K. F. AusTEN. 1973. Alternative complement pathway: factors involved in cobra venoma factor (Co VF) activation of the third component of complement (C3). J. Immunol. 110: 128. 9. VOGT, W., L. DIEMINGER, R. LYNEN, and G. SCHMIDT. 1974. Alternative pathway for the activation of complement in human serum: Formation and composition of the complex with cobra venom factor that cleaves the third component of complement. Hoppe-Seyler's Z. Physiol. Chern. 355: 171. 10. BRADE, V., L. DIEMINGER, G. SCHMIDT, and W. VOGT. 1976. Incompatibility between C 3 band B of guinea pig and man and its influence on the titration of alternative pathway factors f> and B in these two species. Immunology 30: 171. 11. MAURER, H. R. 1968. Disk-Elektrophorese; p. 42. Walter de Gruyter, Berlin 1968. 12. DAMES, 'V., and H. R. MAURER. 1974. Simultaneous preparation for electrophoresis of a large number of micro polyacrylamide gels with continuous concentration gradients. In: Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel. (Ed. by R. C. ALLEN and H. R. MAURER), p. 221. Walter de Gruyter, Berlin, New York 1974.

Purification and Some Properties of Factor

D . 243

13. BRADE, V., A. NICHOLSON, D. BITTER-SUERMANN, and U. HADDING. 1974. Formation of the C 3-cleaving properdin enzyme on zymosan: Demonstration that factor D is replaceable by proteolytic enzymes. J. Immunol. 113: 1735. 14. BRADE, V., A. NICHOLSON, G. D. LEE, and M. M. MAYER. 1974. The reaction of zymosan with the properdin system: Isolation of purified factor D from guinea pig serum and study of its reaction characteristics. J. Immunol. 112: 1845. 15. DIERICH, M. P., U. HADDING, W. KONIG, M. LIMBERT, H. U. SCHORLEMMER, and D. BITTER-SUERMANN. 1974. Factor D in the alternate pathway of complement activation: Purification, physiological characterization and functional role. Immunochemistry 11: 527. Dr. LOTHAR DIEMINGER, Max-Planck-Institut fUr experimentelle Medizin, Hermann-Rein-Stra.l3e 3, D - 3400 Gottingen.