Monoclonal antibodies to human plasma Protein X alias complement

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Protein X alias complement S-protein was isolated by dissociation from purified ... characterize the protein in human serum and plasma. In plasma, Protein X ...
Bioscience Reports 5, 343-352 (1985) Printed in Great Britain


M o n o c l o n a l a n t i b o d i e s to human plasma P r o t e i n X alias c o m p l e m e n t S - p r o t e i n Dieter 3ENNE, Ferdinand HUGO and Sucharit BHAKDI Institute of Medical Microbiology, University of Giessen, Schubertstrasse i, D-6300 Giessen, Federal Republic of Germany (Received 15 April 1985)

Protein X alias complement S-protein was isolated by dissociation from purified XCSb-9 (fluid-phase terminal C5b-9) c o m p l e x e s with 250 mM d e o x y c h o l a t e and subsequent sucrose density gradient centrifugation and SephacryJ gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immunoprecipitate of ~2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of Oh-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monocJonal a n t i - P r o t e i n X a n t i b o d i e s . SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as d i s c e r n e d by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500-700 pg/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades. A plasma protein, originally termed complement 'S-protein' (Podack & MiJller-Eberhard, 1979), appears to inactivate nascent, fluid-phase C5b-9 complexes by firm binding to these molecules with generation of cytolytically inactive complement complexes (Kolb & Mtlller-Eberhard, 1975). This process may represent the last regulatory step in the complement cascade (Bhakdi & Tranum-3ensen, 1983). Further studies



on the biochemistry and function of this plasma protein have been impeded by the difficulties encountered in a t t e m p t s to isolate it from plasma. Following the development of a simple method for isolating the fluid-phase complement complex (Bhakdi & Roth, 19gl), we have t h e r e f o r e r e s o r t e d to an a l t e r n a t i v e procedure for purifying the 'S-protein' which involves dissociation of the protein from C5b-9 by high concentrations of deoxycholate (Podack & Mi~ller-Eberhard, 1980). The purified protein has successfully been used as an immunogen and we report some unusual features disclosed by application of poly- and monoclonal antibodies in electroimmunoassays. Because the protein has been found to fulfil a second function in the coagulation cascade, we have proposed that it provisionally be re-named plasma Protein X (3enne et al., 1985). Materials

and Methods


of protein

X f r o m XC5b-9

XCSb-9 was i s o l a t e d from inulin-activated serum as described elsewhere (Bhakdi & Roth, 1981). Solutions containing 0.6-1.2 mg/ml protein were made 250 mM in deoxycholate (DOC) through addition of solid detergent, and incubated in the presence of 2 mM PMSF for 60 min, 37~ Four ml aliquots were applied to linear sucrose density gradients containing 6.25 mM DOC (10-#0% w/w sucrose in 10 mM Tris, 50 mM NaCI, 7.5 mM NAN,, pH 8.i) and centrifugation performed at 250 000 g x 3 h at #~ in a Beckmann v e r t i c a l rotor type VTi-50 (#0 ml total gradient volume). Twenty equal fractions were c o l l e c t e d from t h e b o t t o m of the tubes and aliquots applied in SDS-PAGE analyses. Fractions containing Protein X were pooled, diluted with # vols. 10 mM Tris, 50 mM NaCI, pH 8.1, and concentrated to approx, one-fifth of the original XCSb-9 volume (Amicon PM 10 membranes). The protein samples were then chromatographed at #~ over a Sephacryl S-300 column (1 x 60 cm, Pharmacia, Uppsala) equilibrated in the same buffer. The flow rate was # ml/h, and 2 ml fractions were collected. This step removed the detergent from the protein and was essential for immunological renaturation. Purified P r o t e i n X was finally concentrated to approx. #00-600 pg/ml and stored frozen at -20~ Rabbit antisera and monoclonal antibodies

Polyclonal antisera were raised in rabbits following the immunization schedule of Harboe and Ingild (1973). Initial injections were with 100 IJg of protein per animal, and booster injections performed with 30-50 pg of protein. Monoclonal antibodies

Female balb/c mice were given 5 x 100 pl Protein X in complete F r e u n d ' s a d j u v a n t ( l : l v / v ) subcutaneously at different sites for primary immunization. They were b o o s t e r e d 5 weeks later by subcutaneous injection of 500 pl of antigen mixed 1:1 with incomplete Freund's adjuvant. A third booster series was given intraperitoneally 5 weeks later without adjuvant, and the mice sacrificed 3-# d t h e r e a f t e r .



Myeloma cells were cultured in RPMI 1640 medium supplemented with 10% i n a c t i v a t e d f e t a l c a l f s e r u m ( B i o c h r o m , Berlin), 1% glutamine, 5000 U/ml penicillin and 5 mg/ml st rept om yci n and 0.02 mM 2 - m e r c a p t o e t h a n o l in a 7% CO 2 atmosphere at 37~ Myeloma cells from 12 petri dishes (9 cm d i a m e t e r ) grown to half confluence were admixed with cells from one spleen in s e r u m - f r e e medium and p e l l e t e d by c e n t r i f u g a t i o n at 12000 r.p.m. The fusion solution consisted of 50% PEG 4000 (Merck, D a r m s t a d t ) , sterilized in RPMI and kept at 37~ in the CO 2 incubator for one day. 2 ml of this solution was added to the cell pellets, which were suspended by gentle pipetting for 2 rain. Four ml of prewarmed medium was then added and the agglutinating ceils continuously swirled for tt rain. The fusion solution was then diluted by addition of 16 ml of RPMI. Cells were pelleted and resuspended in 20 ml of myeloma medium. They were distributed over 4 x 96 cups (0.32 cm 2 culture area) of microtrays containing macrophages from two mice (obtained by peritoneal lavages with 0.34 M sucrose solution). On the next day and every second or third day t h e r e a f t e r ) half of the medium was replaced by HAT-medium (0.04 x 10-2 mM aminopterin, 1.6 x 10 -2 mM thymidine, 0.1 x 10 -2 mM hypoxanthine (in myeloma m e d i u m ) ) . Hybridoma ceils were cloned thrice by limiting dilution on a feeder layer of macrophages in microculture trays. Ceils that were to be seeded were viewed and numbered under the microscope in a Terasaki plate (Nunc GmbH, Wiesbaden). Positive clones were cultured in 157 ml bottles and culture supernatants were collected. Hybridoma cells from two densely grown culture flasks were intraperitoneally injected in balb/c mice p r e - t r e a t e d with 0.5 ml of pristane, and ascites fluid co llected 2-5 weeks t h e r e a f t e r . Screening assays. R o u t i n e s c r e e n i n g was p e r f o r m e d using m i c r o - E L I S A a s s a y s with p l a t e s c o a t e d with purified Protein X; development was with peroxidase-labelled second antibodies. Definitive i d e n t i f i c a t i o n of a n t i b o d y specificities was p e r f o r m e d by e l e c t r o immunoassay-immunoblotting (EIA-IB) as described e l s e w h e r e - ( B h a k d i et al., 1985). In the later stages of this work, a simple line-immunoe l e c t r o p h o r e s i s - i m m u n o b l o t t i n g screening assay was used. Human s e r u m was a p p l i e d in line i m m u n o e l e c t r o p h o r e s i s against specific antibodies to antithrombin-III (AT-III; Atlantic Antibodies, Scarborough, Memphis). The r e s u l t i n g line immunoprecipitate, which has been shown to contain not only antithrombin-III, but also complexed Protein X, was solubilized in SDS and e l e c t r o b l o t t e d onto nitrocellulose. The filter paper containing the blotted proteins was then sectioned into 0.5 cm wide strips and used to test the r e a c t i v i t y of individual antibody clones for protein X - r e a c t i v i t y as described elsewhere (Bhakdi et al., 1985). O n e - and t w o - d i m e n s i o n a l SDS-PAGE/immunoblotting were performed as described elsewhere (Bhakdi & Tranum-3ensen, 1982; Bhakdi et al., 198/4). Blots were ceveloped with peroxidase-labelled rabbit anti-mouse IgG (Dakopatts, Copenhagen). Plasma concentrations of Protein X in 10 h e a l t h y d o n o r s w e r e d e t e r m i n e d by r o c k e t - i m m u n o e l e c t r o p h o r e s i s immunoblotting using purified protein as standard, Protein c o n c e n t r a tions in the standard were determined by amino acid analyses.



R e s u l t s and D i s c u s s i o n Isolation of Protein X from XC5b-9

XCSb-9 p r e p a r a t i o n s were given 230 mM DOC and centrifuged through sucrose density gradients. SDS-PAGE analyses of individual gradient fractions showed that Protein X was selectively and partially dissociated from the complex. The upper gradient fractions containing the protein (Fig. IA) were chromatographed over Sephacryl S-300 to remove detergent. This step led to recovery of the protein (Fig. 1B) in immunologically intact form in overall yields of 23-30%. The amino acid composition of Protein X is given in Table 1. The results a r e in good a g r e e m e n t with data of Podack and Mi~ller-Eberhard (1979) for Protein X purified from plasma. The absorbance at 280 nm of a 1 mg/ml protein solution (A280, 1 cm) was 1.7. Electroimmunoassays with polyclonal rabbit antibodies

P u r i f i e d P r o t e i n X y i e l d e d a single immunoprecipitate of c~e l e c t r o p h o r e t i c mobility upon crossed immunoelectrophoresis against specific rabbit antiserum (Fig. 2A). Surprisingly, however, we always observed the development of 3-10 immunoprecipitates upon analysis of whole human serum with this antiserum (Fig. 2B). Thus, the Protein X preparations still contained strongly immunogenic contaminants. In

Fig. I. Purification of Protein X from fluid-phase XC5b-9 (formerly SC5b-9) complement complexes. XCSb-9 (A: left gel) was treated with 250 mM DOC. The sample was centrifuged through a linear sucrose density gradient, twenty fractions were collected, and aliquots applied to SDS-PAGE (A: lanes 1-20). Protein X was selectively and partially dissociated from the XCSb-9 complex to be recovered in the upper fractions (15-19) of the gradient. Fractions 16-19 were pooled and chromatographed over Sephacryl S-300 to remove detergent. Purified Protein X recovered from the column (gel B) exhibited the typical diffuse staining pattern in SDS-PAGE.


Table I.




Amino acid composition of Protein X

Amino acid

Residues/100 residues

Asp Thr Ser Gln Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg

12.1 5.4 7.6 13.4 6.5 7.0 6.1 2.1 5.0 i. 7 2.6 8.2 3.1 5.2 5.9 2.2 5.9

Fig. 2. Crossed immunoelectrophoresis of isolated Protein X (plate A) and human serum (plates B, C) developed with a polyclonal rabbit antiserum to Protein X. The isolated protein (A) yielded a symmetrical immunoprecipitate of e-electrophoretic mobility with this antiserum. However, 8-10 precipitates were observed when serum was applied (B, C). In plate C, purified Protein X was incorporated into a 1 cm wide intermediate gel; plate B (control) contained a blank gel. The line immunoprecipitate (curved arrow, plate C) representing Protein X fused with a major, double-peaked immunoprecipitate (arrows), provisionally identifying the latter as serum Protein X.




order to provisionally identify Protein X, a crossed-line immunoelectrophoresis (Kr~ll, 1973) was performed with incorporation of purified protein in an intermediate gel (Fig. 2C). This resulted in fusion and elevation of a single, double-peaked precipitate corresponding to serum Protein X onto the line precipitate (plate 2C). EIA-IB with monoclonal antibodies

Five monoclonal antibodies were obtained against Protein X, three of subclass IgG-1, one of subclass IgG-2b, and one of not clearly identifiable subclass. All bound to the protein in crossed-immunoe l e c t r o p h o r e s i s / i m m u n o b l o t t i n g analyses. In these assays purified P r o t e i n X was first immunoprecipitated in crossed immunoelectrophoresis with the rabbit antiserum (as in Fig. 2A). The precipitate was dissolved in SDS, etectroblotted, and reacted with the monoclonal antibodies. The blots were developed with peroxidase-labelled second a n t i b o d i e s to mouse immunoglobulins. Fig. 3A shows a positive immunoblot obtained with a monoclonal anti-Protein X antibody. We next used the monoclonal antibodies to analyse the immunoprecipitation behaviour of Protein X in serum (Fig. 3C) as opposed to plasma (Fig. 3B). These analyses showed that whereas the protein yielded a symmetrical immunoprecipitate of ~-electrophoretic mobility when analysed in plasma, a double-peaked precipitate formed when serum was applied as antigen. These results confirmed the observations of Fig. 2 and demonstrated a partial change in the physical state of the protein that occurred during blood coagulation. As reported in a separate communication, the slowly moving immunoprecipitate has been i d e n t i f i e d as a complex between Protein X and serum antithrombin III (3enne et al., 1985). Here the co-precipitation of Protein

Fig. 3. Crossed immunoelectrophoresis combined with e l e c t r o b l o t t i n g with monoclonal anti-Protein X antibodies. A: Purified Protein X. B: EDTA-plasma (3 ~I). C: Whole serum (5 ~i). In the first stage, the samples were i m m u n o e l e c t r o p h o r e s e d against the polyclonal rabbit anti-Protein X antiserum of Fig. 2. The precipitates were subsequently dissolved in SDS and electroblotted o n t o nitrocellulose. The blots were finally developed with monoclonal anti-Protein X antibodies. Note the presence of a symmetrical Protein X immunoprecipitate in plate B (EDTA-plasma) as opposed to the double-peaked precipitate in plate C (serum).



X with serum antithrombin-III is demonstrated in another manner. Isolated AT-III (obtained from Behringwerke, Marburg) or whole serum was s u b j e c t e d to crossed immunoelectrophoresis against anti-AT-III antibodies. C o n d i t i o n s were adjusted such that similar immunoprecipitates developed in both plates. The precipitates were then dissolved in SDS and electro-immunoblotted using monoclonal antiP r o t e i n X antibodies. Fig. 4 depicts the results showing positive staining for Protein X in the AT-III precipitate derived from serum, but entire absence in the precipitate derived from purified plasmaderived AT-III, which contained no complexed Protein X. The above observation was exploited for development of a simple screening assay which permitted definitive identification of monoclonal anti-Protein X antibodies. For this purpose, serum was subjected to line i m m u n o e l e c t r o p h o r e s i s (Krgll, 1973) against anti-AT-III. The ensuing line immunoprecipitate (Fig. 4C) containing the co-precipitated Protein X was electroblotted, and the filter paper then sectioned into 0.5 cm wide strips. Cell culture supernatants or ascites fluids were applied to individual strips, and specific anti-Protein X clones were identified through their positive reactions (Fig. 4C). Plasma levels of Protein X in i0 healthy adults were determined by rocket immunoelectrophoresis-immunoblotting, using purified protein as the standard, and found to be of the order of 500-700 tJg/ml. This e s t i m a t e agrees with the values given by Podack and Mflller-Eberhard (1979). SDS-PAGE immunoblotting

SDS-PAGE immunoblots developed with a mixture of 4 monoclonal anti-Protein X antibodies consistently led to the generation of multiple diffuse protein bands in the approx. Mr region of 80-90 000 (Fig. 5). A d d i t i o n a l , faint staining was observed in higher molecular weight regions. No differences were noted between plasma and serum, and the pattern also remained unaltered a f t e r inulin activation of serum with formation of XC5b-9 (Fig. 5A). Thus, Protein X does not appear to be cleaved to lower molecular weight entities during the process of blood coagulation or during XC5b-9 formation. Reduction with 10 mM d i t h i o t h r e i t o l led to sharpening and more intensive staining of the protein in the 80 K region, and concomitant generation of a lower molecular weight polypeptide of 15 K. These results were confirmed and extended by two-dimensional SDS-PAGE/immunoblotting (Fig. 5B,C). First dimension samples were electrophoresed under non-reducing conditions, followed by second dimension electrophoresis with disulfide bond cleavage. We consistently observed the generation of a major polypeptide of approx. 80 000 daltons also deriving from high m o l e c u l a r w e i g h t m a t e r i a l in the f i r s t d i m e n s i o n gels. Additionally, we found faint but distinct staining of a low molecular weight polypeptide (approx. 15 K, Fig. 5C) and microheterogeneity in the 80-90 000 dalton region. The collective results indicated that Protein X exists in heterogeneous form in plasma, and that disulfide bonds play a role in stabilizing its structure. A low molecular weight polypeptide of 15 K is cleaved from the native protein by dithiothreitol; whether this cleavage product is generated as the result of i n t r a m o l e c u l a r ' n i c k i n g ' of Protein X, or whether the protein is actually primarily composed of two disulfide-bonded polypeptide chains



Fig. 4. A: Crossed immunoelectrophoresis followed by i m m u n o b l o t t i n g of purified antithrombin-III developed with monoclonal anti-Protein X antibodies. In the first stage, antithrombin-III was precipitated with anti-AI-III in conventional crossed immunoelectrophoresis. The precipitate was dissolved in SDS, electroblotted, and subsequent incubation of the nitrocellulose blot with monoclonal anti-Protein X antibodies yielded an entirely blank immunoblot. Thus, AT-III isolated from human plasma contained no Protein X. B: In contrast, AT-III derived from human serum was precipitated with anti-AT-III and the immunoprecipitate was shown to contain Protein X. C: Line immunoelectrophoresis combined with immunoblotting for identification of anti-Protein X antibody clones. Human serum was e l e c t r o p h o r e s e d in a line immunoelectrophoresis against anti-AT-III. The precipitate was electroblotted, the filter paper then sectioned into 0.5 cm wide strips, and individual antibody clones tested for their reactivity. In this manner, anti-Protein X clones (strips y, z) were easily distinguishable from other clones (strip x), which yielded blank immunoblots.






Fig. 5. A: SDS-PAGE immunoblotting of (a) EDTA-plasma; (b) serum; (c) inulin-activated serum developed with monoclonal anti-Protein X antibodies. Note the development of diffuse bands (arrows) in the M r region 80-90 000 and additional faint staining in higher molecular weight regions. Samples were not reduced prior to electrophoresis. B and C: Two-dimensional SDS-PAGE immunoblotting of EDTA-plasma. First dimension SDS-PAGE (left to right) under non-reducing conditions. Second dimension electrophoresis on 12.5% gels (top to bottom) under non-reducing (plate B) or reducing conditions (plate C). Note the generation of s t r o n g l y staining spots exhibiting pronounced m i c r o h e t e r o g e n e i t y and the presence of a weakly staining moiety of approx. 15 K (arrow) in plate C. The major 80 K moiety and the small 15 K polypeptide were also generated from high molecular weight material stemming from the origin of the first dimension gel. Thus, polymeric aggregates of Protein X appear to be present in plasma; their weak staining in plate B and gels a-c is probably due to poor transferability from the SDS-gels by electroblotting in the absence of disulfide reducing agents.




requires future clarification. It is noteworthy that Protein X present in isolated XC5b-9 complexes was reported to be cleaved by dithiothreitol into a major #0 000 dalton subunit (Bhakdi & Tranum-3ensen, 1982). The present data would indicate that this was due to secondary nicking of the protein occurring during the isolation procedure. The m e t h o d s d e s c r i b e d in this paper a p p e a r well suited for p u r i f i c a t i o n of Protein X in immunologically intact form. In an accompanying paper, we have shown that the isolated protein still binds to AT-III/thrombin complexes to exert a net thrombin-protective function (3enne et al., 1985). The availability of poly- and monoclonal antibodies to the protein should facilitate and promote further studies on this novel and interesting plasma component. Acknowledgem ents We thank Margit Pohl and Marion Muhly for outstanding technical assistance and M. Wiesner (Max-Planck-Institute, Freiburg) for kindly performing the amino acid analyses. This work was supported by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Bh 2/1-5) and the Fonds der Chemischen Industrie. References Bhakdi S & Roth M (1981) J . Immunol. 127, 576-582. Bhakdi S & Tranum-Jensen J (1982) Mol. Immunol. 19, 1167-1176. Bhakdi S & Tranum-Jensen J (1983) Biochim. Biophys. Acta 737, 343-373. Bhakdi S, Muhly M & Fussle R (1984) Infect. Immun. 46, 318-323. Bhakdi S, Jenne D & Hugo F (1985) J. Immunol. Methods, in press. Harboe N & Ingild A (1973) Scand. J. Immunol. 2, Suppl. I, 161-164. Jenne D, Hugo F & Bhakdi S (1985) Thromb. Res., in press. Kolb WP & MUller-Eberhard HJ (1975) J. Exp. Med. 141, 724-725. Podack ER & MUller-Eberhard HJ (1979) J. Biol. Chem. 254, 9908-9914. Podack ER & MHller-Eberhard HJ (1980) J. Immunol. 124, 1779-1783.