bactericidal factor, Ra reactive factor. complement by a proteinase in ...

0022-1 767/93/1502-571$02.00/0 The Journal of Immunology Copyright 0 1993 by The American Association of Immunologists

Vol 150, 571 -578, No. 2, January 15, 1993 Printed in U.S.A.

Activation of the C4 and C2 Components of Complement by a Proteinase in Serum Bactericidal Factor, Ra Reactive Factor' Yue-Hua Ji,** Teizo Fujita,' Hiromi Hatsuse,* Akiyoshi Takahashi,** Misao Matsushita,' and Masaya Kawakami3* *The Department of Molecular Biology, School of Medicine Kitasato University, Sagamihara, Kanagawa 228, Japan; and 'Department of Biochemistry, Fukushima Medical College, Fukushima, Japan

ABSTRACT. Ra-reactive factor (RaRF) is a C-dependent bactericidal factor that bindsspecifically

to LPS of Ra

chemotype strains of Salmonella and kills the bacteria by triggering the C cascade. In the present study, we investigated the components of mouse RaRF that activate C4 and C2. The RaRF bound to LPS-coated E, and activated the C4 on the surface of E, causing theC4 to bind to the cells. Diisopropyl fluorophosphate (DFP) bound to RaRF and inhibited its ability to activate C4 and C2. Cleavage of the a-chain of C4 by RaRF generated a polypeptide with a size similar to that of the a'-chain of C4b, which is known to be a product ofthe cleavage of C4 by C1 s subcomponent of C1. A fraction with the ability to activate C4 and C2 was separated from RaRF by gel-permeation chromatography in the presence of EDTA and acetonitrile. This fraction contained a DFP-binding polypeptide with an apparent m.w. of 100,000. This polypeptide is not the C1 s in mouse C1 because the sizes of this polypeptide and of the fragments produced by its reduction weredifferent

from those of DFP-binding

proteinases in mouse C1. These results indicate that mouse RaRF contains a C1 s-like serine proteinase that is capable of activating C4 and, probably, C2. Journal of Immunology, 1993, 150: 571.

R

aRF4 is the name given to members of a group of bactericidal factors that are present in sera of a wide variety of vertebrates from mammals through fishes ( 1 4 ) . This factor binds specifically to the Ra and R2 core polysaccharides of LPS (1, 4) which are Received for publication April28, 1992. Accepted for publication October26,

1992. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Sectlon 1734 solely to indicate this fact.

'

This work was supported by grants from Ministry of Culture, Education and Science, Japan (63570200 and 63570277) and from The Terumo Life Science Foundation. Present address: Y.-H. li, Department of Microbiology, TheShanghai Second Medical College, Shanghai, China; A. Takahashi, LaboratoryofMolecular Endocrinology, School of Fishery Sciences, Kitasato University, Sagamihara, Kanagawa, 228 japan.

shared by many strains of enterobacteria, such as Salmonella species and Escherichia coli ( 5 , 6). The RaRF kills the bacteria by activating C (2, 4).Using hemolytic system, we have demonstrated that the C4 and C2 components of C are responsible for triggering the activation of the C cascade (7). Moreover, the C 1 component was found to be unnecessary for this reaction. Mouse RaRF is a protein complex with a m.w. of approximately 310,000, which is composed of high and low m.w. components (8). Upon reduction, the high m.w. component generates two kinds of 28-kDa polypeptides, P28a and P28b, and the low m.w. component produces 29-kDa 1 m M CaCI,; CVB.Veronal-bufferedsalineContaininggelatin: EDTA-CVB, Veronal-buffered saline containing gelatin and EDTA; MCVB, Veronal-buffered saline of low ionicstrength containing 2.3% mannitol, 0.1 5 mM CaC12, 1 .O mM MgCI2, and 0.1% gelatin, p H 7.3 pm 0.07; ""T2, oxidized C2: ELPS, sheep erythrocytescoated with LPS from Ra bacteria; ELPS-RaRF,ELPS sensitized with RaRF; EA, sheep erythrocytes sensitized with antibody; C4D, C4-deficient guinea pig serum; C-EDTA, guinea pig serum diluted 1/50 with EDTA-MCVB as a source of C3-C9: EDTA-MCVB, MCVB containlng 10 m M EDTA but no Ca and M g salts.

% Address correspondence and reprint requests to Dr. Masaya Kawakaml, Department of Molecular Biology, School of Medicine, Kitasato University, Sagamihara, Kanagawa, 228 Japan.

Abbrevlations used in this paper: RaRF, Ra-reactive factor; DFP, diisopropyl fluorophosphate; TBS, 20 m M Trls-HCI, p H 8.0, containing 130 m M NaCl and

571

5 72

C4/C2-ACTIVATING COMPONENT OF BACTERICIDAL FACTOR, RaRF

and 70-kDa polypeptides and others (8,9). We have found that the high m.w. component no longer activates c after it has been separated from thelowm.w. component, although it retains the ability to bind specifically to the Ra determinant (8). In the present study,we examined the factors required for the activation of the C4 and C2 components of C. The results indicate that a serine protease capable of activating these components is present in the low m.w. component of the RaRF protein complex. The present and previous results suggested that the RaRF complex is similar to the C1 component of C with respect to both function and polypeptide composition.

Materials and Methods Preparation of RaRF

RaRF was prepared from serum of ICR mice by adsorption onto bacterial cells of an Ra chemotype strain, rfb388, of Salmonella typhimurium and elution with 10% N-acetylD-glucosamine in TBS (7). The C l and Ig were removed from the eluate (3 ml) by passage of this preparation through a column of rabbit antibodies against mouse Ig bound to Sepharose 4B (0.9 X 16 cm; Pharmacia, Uppsala, Sweden). The purified material was condensed by ultrafiltration, with a microconcentrator (Centricon; Amicon, Beverly, MA) or by precipitation with 5 volumes of chilled acetone. Buffers, C components, and reagents

Isotonic GVB and EDTA-GVB were prepared as described elsewhere (10). MGVB and EDTA-MGVB were also used (7). RaRF was removed from all sera used as sources of C by adsorption with Ra bacteria (7). Human C4 (1 l), C2 (12), and "'YC2 (13), and guinea pig C1 and C4 (14) were prepared as described previously. C-EDTA was used as a source of C3-C9. C4D was obtained from guinea pigs with a genetic deficiency of C4. A fraction of functionally pure C1 wasprepared from mouse and human serum by precipitation in a medium of low ionic strength and subsequent gel-permeation chromatography on Sepharose 6B, as described elsewhere (7, 15). Cellular intermediates and hemolytic assays

ELPSand ELPS-RaRF were prepared as described (7). Antibody-sensitized E were prepared by the standard method (10). EACl and EAC14 cells were prepared as described previously (7, 10). Methods for the titration of the hemolytic activities of RaRF (7), C1, C4, and C2 (16) have also been described previously. The activity is expressed in terms of site-forming units, calculated from the Z value of the hemolysis.

C4 and C2 consumptions

Samples in 50 pl of MGVB were mixed with 4 U of human C4 or 12.5 U of human "'YC2 in 50 p1of MGVB. After incubation for 20 min at 30"C, the remaining activity of C4 and C2 was measured by the system of sensitized E (EA) as described previously (10). Sensitivity to DFP of RaRF and C1

One milliliter of ELPS-RaRF or EACl (1.5 X 108/ml)was mixed with the same volume of MGVB containing 10 mM DFP and incubated at 30°C. After various intervals of time, aliquots of cells were withdrawn, washedwith MGVB, and incubated with excess U of guinea pig C4 for 20 min at 30°C. The cells were washed and resuspended with 100 pl of MGVB. The remaining activity of RaRF or C1 on the cells was determined by further incubation with 1 plof C4D in 100 plof MGVB.In another series of experiments, an aliquot of DFP-treated cells was washed with MGVB, incubated with excess U of human C4 in 100 pl of MGVB for 20 min at 30°C. After washing and resuspending of cells with MGVB, the RaRF or C 1 activity was determined by incubation with 20 U of "'YC2 in 100 plof MGVB and then with 1.2 mlofC-EDTA,as described elsewhere (7). C4-binding tests and structural analysis of cell-bound

['2511c4 Highly purified human C4 was labeled with 1251 (Amersham International, Amersham, U.K.) by use of Iodogen (Pierce Chemical Co., Rockford, IL), andunbound IZ5I was removed by gel filtration through Sephadex G25 and subsequent extensive dialysis (16). The sp. act. was 4.7 X lo5 cpdpg. One hundred microliters of suspension of ELPS, ELPS-RaRF, EA, or EACl cells (1.5 X 108/ml) were incubated with 100 p1 of radiolabeled C4 (15 pg/ml) for 15 min. After washing twice the cells withMGVB containing 0.1% BSA, the amount of bound C4 were determined by measuring radioactivity. The cellular intermediates, carrying IZ5I-labeledC4, were lysed in 50 mM Tris-HC1, pH6.8, containing 2% SDS, 6 M urea, 10% glycerol, and10% ME (16). After removal of insoluble materials by centrifugation, samples were analyzed by SDS-PAGE. DFP-binding tests

Sample protein (20 pg) was incubated in 50 p1 of TBS containing 10 mM EDTA for 30 min at 37°C. The incubated sample wasmixedwith 10 pCi of [1,3-3H]DFP (4.4 Ci/mmol; NEN Research Products, Boston, MA), and incubated overnight at 4°C. The sample protein was precipitated andwashedwith cold acetone to remove free

5 73

Journal of immunology

[3H]DFP. Radioactivity in the protein fraction was determinedin a liquid scintillation counter (model LSC602; Aloka, Tokyo, Japan).

i'o

c 0

PAGE and autoradiography

SDS-PAGEwas performed by the methodof Laemmli (17). Unreduced samples were applied to a gradient slab gel (4-12%).In other cases, samples were reduced by incubation for 3 min at 90°C in Laemmli's sample buffer supplemented with 100 mM dithiothreitol or 6 M urea plus 10% ME, and applied to a 10% slab gel (9). After electrophoresis, gels were soaked inAmplify (Amersham Japan Co., Tokyo,Japan), stained with Coomassie brilliant blue G250, and dried. Autoradiography was carried out by exposure of the dried gel to Fuji x-ray film RX (Fuji Film Co., Tokyo, Japan) at -70°C for 20 to 72 h. Gel-permeation chromatography

Samples dissolved in 100 p1 of a mixture of 1 volume of acetonitrile and 9 volumes of 500 mM ammonium acetate containing 5 mM EDTA, pH 6.7, were loaded onto a column for HPLC (7.8 X 30 cm; TSK G3000SW, Tosoh Co., Tokyo, Japan) and eluted with the same medium at a flow rate of 1.0 mVmin at 22°C. Fractions (0.8 ml each) obtained after several repeats of this chromatographic procedure were pooled. Part of the pooled fraction (350 pl) was subjected to precipitation of proteins with TCA and used for the analysis of proteins by SDS-PAGE.After removal of acetonitrile from the remainder of the fraction by evaporation, the protein was precipitated with cold acetone in the presence of 50 pg of bovine y-globulin, as carrier, and used for the measurement of the C4-consuming activity and for the PAGE analysis of DFP-binding polypeptides.

Results Consumption of C4 and C2 by RaRF

TheC4 component of C was incubated with various amounts ofRaRF, and the residual activity of C4 was assayed in the EA hemolytic system. The activity of the C 1 component of C was also examined as a control. As expected, C1 caused a reduction in the C4 activity, as shown in Figure 1A. It was found that the RaRF also reduced C4 activity in a dose-dependent manner. By calculation of the 50% hemolytic doses before and after incubation with RaRF, it was estimated that approximately 6.8 U of C4 out of a total of 7.8 U of C4 in the reaction were consumed by 3 ngofRaRF. Approximately 11.5 U of C2 were also consumed by 300 ng of RaRF, whereas theconsumption of C2 by C1 was incomplete (Fig. 1B).

1

10

FIGURE 1. Consumption of C4 and C2 by RaRF in the fluid phase. A, RaRF and mouse C1 were incubated in TBS containing 10 mM EDTA for 3 0 min at 37°C. Various amountsof RaRF or C1 (A)were mixed with 7.8 U of guinea pig C4 in 100 pi of MGVB. After incubation for 20 min at 30"C, C4 activity remaining in the mixture wasassayed with EA and C4D. 8,various amounts of RaRF (O),or C1 (A)were mixed with 12.5 U of human " " T 2 in 100 pI of MGVB. After incubation for 20 min at 30"C, C2 activity remaining in the mixture was assayed with EACl4 cells and C-EDTA.

(a),

Cleavage of C4 molecules by RaRF

Purified C4 was incubated with various amounts of RaRF or C 1s in a liquid medium, and the products formed by cleavage of C4 were examined by SDS-PAGE. As shown in Figure 2, the treatment of C4 with C l s resulted in the cleavage of the a-chain, producing the a'-chain, being consistent with the previous report (18). RaRF also produced a polypeptide with a m.w. the same with or very similar to that of the a'-chain, and the amount of product was dependent upontheamount of RaRF added. The amounts of RaRF and C l s required to cleave 50% of C4 were roughly estimated to be 8 and 1 ng, respectively. The m.w. of RaRF and C l s are assumed to be 310,000 and 85,000, respectively. Therefore, it seems thatthe efficiency of cleavage by RaRF is not lower than one-fourth of C1 s in terms of molar concentrations because the amount of C4-activating factor in the RaRF complex is less than 40% as deduced from the intensities of bands after SDS-PAGE, as shown below. Next, we investigated whether C4 boundto ELPSRaRF. As controls, ELPS, EA, and EACl cells were set up. The rates of binding of radiolabeled C4 to ELPS-RaRF and to EACl were 0.3% and4.3%, respectively (not shown). No significant amounts of C4 were bound to ELPS and EA. Asseen in the autoradiograph of a gel after SDS-PAGE shown in Figure 3, incubation of EACl with C4 generated a protein with a mobility similar to that of the a'-chain of C4. In contrast, certain proteins of high m.w.were generated when ELPS-RaRF was incubated with C4, although no band corresponding to the a'-chain appeared. It is likely that the radioactive bands corresponding to proteins of high m.w. represent complexes

574

C4/CZ-ACTIVATINGCOMPONENTOFBACTERICIDALFACTOR,RaRF

1 2

3 4 5 6

7 8 9 10 1 1 12 13 Marker

a chain, a ' chain@ chain(

94K

67K

43K y

chain30K 2UK

FIGURE 2. Cleavage of C4 by RaRF in the fluid phase. RaRF or human C1 s at various concentrations (0.15 to 15 pg/ml) was incubated with a constant concentration of C4 (1 33 pg/ml) for 30 min at 30°C in 100 pI of MGVB. Proteins were analyzedby 10% SDS-PACE under reducing conditions. Lanes 1 , 7, and 13, C4 incubated without RaRF and C1s; lanes 2 to 6, with 0.1 5, 0.5, 1.5, 5, and 15 pg/rnl RaRF; lanes 8 to 12, with 0.15, 0.5, 1.5, 5, and 15 pg/rnl Cls. M, m.w. markers. The a-chain ofC4

was cleaved in the a'-chain byRaRF as well as by Cls. caused byan ester-like linkage of the a'-chain of C4 to LPS. From these results, it is clear that RaRF contains a proteinase that is capable of cleaving the a-chain of C4 at the same or similar site to that cleaved by CIS, and that C4b binds to ELPS-RaRF, probably through an ester-like linkage. Reactivity of RaRF with DFP Sensitivity to DFP of the predicted proteinase in the RaRF complex was examined. ELPS-RaRF were incubated with DFP for various lengths of time. After washing the cells, the ability of RaRF to activate C4 was assayed. This ability decreased markedly during incubation withDFP for I O min or longer as shown in Figure 4. The C1 that bound to EA was also sensitive to DFP. The decreases in the rate of lysis of ELPS and of EACl are not caused by DFP that remains in the erythrocytes after washing because treatment of EA with DFP before addition of C1 had no effect on the CI-induced lysis (data not shown). DFP-binding activity of RaRF was investigated by incubating RaRF with ['HIDFP in a liquid medium at pH 5.5. After incubation of RaRF with 95 nmol of DFP for 120 min, approximately 0.28 nmol of DFP was bound to 310 pgofRaRF,which corresponds to about 1 nmol of protein (data not shown). Under the same conditions, 0.15 nmol of DFP bound to 1 nmol of humanC I s. These results

indicate that the mouse RaRF complex contains a serine proteinase similar to human CIS. It is very likely that this proteinase is responsible for the activation of C4, because the CCactivating ability of RaRF is sensitive to DFP. Size of the DFP-binding polypeptide in RaRF After treatment of the RaRF complex with['HJDFP, polypeptides in reduced and unreduced RaRF were separated by SDS-PAGE. Mouse CI was used as a control. As seen in the autoradiograph shown in Figure 5 , the unreduced RaRF gave a major band corresponding to a DFPbinding polypeptides withm.w.of 100,OOO after SDSPAGE. Under reducing conditions, RaRF formed a band of DFP-binding protein of 29 kDa. The CI preparation used is thought to contain both the Clr and CIS because a full function of CI in this preparation was demonstrated by using EA system (7). However, the DFP-treated mouse C 1 generated only a single radioactive band under the present PAGE conditions (Fig. 5 ) although two bands formed within a short distance when the SDS-PAGE was carried out by using 6% gel (not shown). The mobility of the radiolabeled components of C 1 was clearly different from that of the DFP-binding component of RaRF under both sets of conditions. The 100-kDa polypeptide is not a product of the mouseC 1 s because the m.w. of this polypeptide, which is estimated by the PAGE analysis, is higher than that of mouse C 1s. It was demonstrated by SDS-PAGE on

Journal of Immunology

575

Incubatlon T l r (.In)

-7

0.0

1 2

3

FIGURE 3. Binding of C4 to ELPS-RaRF.ELPS,ELPS-RaRF, EA, or EACl cells (1.5 X 107/100 pl) were incubated with 100 pI of radiolabeled C4 (30 pCi). After washing the cells twice the amounts of bound C4 were determined by measuring radioactivity. The cell membranes were lysed with SDS-urea. Under these conditions, LPS, together with the C4 bound to LPS,was solubilized, whereasthe C4 bound to insoluble materials on cell membrane was removed by centrifugation. The solubilized materials were analyzed by SDSPAGE and subsequent autoradiography. About 20,000 cpm of each sample were loaded onto each lane. Lane I , EACl + C4; lane 2, ELPS-RaRF + C4; lane 3, C4 only.

a separate gel that the mobility of the radioactive band of mouse C1 under nonreducing conditions was almost the same with that of human Cls, and that its mobility under reducing conditions corresponded to that of the L chain of human Cl r (not shown). Therefore, it is considered that DFP-binding polypeptides of RaRF and mouse C1 differ from one another in terms of the m.w. of the intact and reduced molecules, whereas those in mouse and humanC 1 are similar. Separation of the proteinase from other components of RaRF

Components of RaRF were separated by gel-permeation chromatography in the presence ofEDTA and acetonitrile. As shown in Figure 6, the peak of C4-consuming activity coincidedwithan OD peak corresponding to100-kDa protein. The C4- andCZconsuming activities of fraction6 in the chromatography were determined quantitatively in an experiment similar to thatfor which results were shown in Figure 1. Liketheunfractionated RaRF, thisfraction consumed C4 and C2 in a dose-dependent manner. Comparison of the 50% inhibition doses offraction 6 and unfractionated RaRF indicated that thesp. act. with respect to consumption of C4 and C2 in fraction 6 were increased 15.8-and5.4-fold,respectively, by the fractionation. In

FIGURE 4. Inactivation of RaRF on ELPS by DFP. A total of 1.5 X 10' of ELPS-RaRF or EACl were incubated with DFP (5 mM) in200 pl of MGVB, pH 7.3, for the indicated lengths of time at30°C.The cells werewashed with MGVB and resuspended in 100 pl of thesame medium. Activities of RaRF and C1 on the cells were assayed by two methods as follows: A, one series of cells was assayed by serial incubations with guinea pig C4 (6.5 U) and C4D (1 pl). B, another series was assayed by serial incubations with purified human C4(2.3 U), O"YC2 (20 U),and C-EDTA.The cells were washed with MGVB after each incubation. ELPS-RaRF incuor without DFP (0); EACl incubated with (A) bated with (0) or without DFP (A).

Unreduced

Reduced I i

I i

e m

100k80k

-

e m

*

e

u

-

0

e

32k-

29k-

-

u

-

FIGURE 5. Analysis of DFP-bound polypeptides in RaRF and C1. Polypeptides of [3H]DFP-treated RaRF and mouse C1 were separated by SDS-PAGE on a 4 to 12% gradient gel under nonreducing conditions (left)and on a 12% gel under reducing conditions (right).Autoradiographs are shown. The estimated m.w. of proteins calculated from the Rf of marker proteins are shown on the left of the gels.

contrast to the enhanced C4- and CZconsuming activities, the capacity for lysing ELPS with the aid of C4, C2, and C-EDTA was completely absent in this fraction (data not shown). This observation suggests that no binding activity is associated with this fraction, probably because ofthe absence of the binding component. SDS-PAGE under nonreducingconditions revealed that

C4/CZ-ACTIVATING COMPONENT RaRF OFFACTOR, BACTERICIDAL

576

E g

OS3lI

0

I

N

- PlOO

c

5.

0.2-

c 2 3 4 5 6c 2 3 4 5 6

A .

-100

n

.r

-" c

76K-

c

0

-

0.1-

.r

50 .? h

53K

7

0

I

I

1 2 3 4 5 6 7 6 9 1 0 1 1

Fractions FIGURE 6. Separation of theproteinase in RaRF by gelpermeation chromatography. RaRF (900 pg) dissolved in 100 pl of 500 mM ammonium acetate containing 5 mM EDTA and 10% (v/v) acetonitrile was loaded on a TSK C3OOOSW column and eluted with the same solution. -, AZRonm; - - -, activity of 300-fold dilutedfractions in terms of consumption of C4 (4 U), expressedas the inhibition of hemolysis ( O b ) .

fractions 5 and 6 contained a DFP-binding, 100-kDa polypeptide (Fig. 7). Under reducing conditions, this fraction gave two bands: a DFP-binding polypeptide of 29 kDa and nonbinding polypeptide of 70 kDa. These results are consistent with those from SDS-PAGE of unfractionated RaRF shown in Figure 5. In addition to the bands of 29,000, 70,000, and 100-kDa polypeptides, several bands of smaller polypeptides were revealed by the PAGE of fractions 5 and 6 under reducing and nonreducing conditions. These polypeptides are probablyproduced by the degradation of native proteinase molecules during the separation by gel-permeation chromatography and DFP-labeling procedures. The results described above indicate that the RaRF complex is composed of at least two kinds of components: one with a m.w. of more than 200,000 and another with m.w. of 100,000. The high m.w. component is an oligomer of 28-kDa polypeptides, P28a and P28b, as demonstrated in a previous study (8). The 100-kDa component (PIOO) is a serine proteinase composed of a DFP-binding 29-kDa and nonbinding 70-kDa polypeptide chains (Fig. 8). These chains are bound by a disulfide bridge, probably like the H and L chains of the activated CIS molecule.

Discussion The following results have been obtained in the previous studies. Mouse RaRF directly activates the C4 and C2 components of C without the participation of the CI (7). The RaRF protein complex is composed of high and low m.w. components. The former component, whichisan oligomer of P28a and P28b, has the activity to bind to the Ra determinant, but not to consume C4 (8). Therefore, it has been assumed that there is a CWC2-activating factor in

-

B 94K67K--

"" "a

43K-

25 K20K-

" ! r

---

"

"29K 'P2P

FIGURE 7. SDS-PAGE of the fractions from gel-permeation chromatography of RaRF. Each eluted fraction indicated in Figure 6 was labeled with ["IDFP, and analyzed by SDSPAGE under nonreducing ( A ) and reducing conditions (B). Left, stained gels; right, autoradiographs. LaneC, unfractionated RaRF; lanes 2 to 6, fractions 2 to 6. Molecular weight of marker proteins are indicted on the left side of the gels.

-

Polysaccharide-binding components P28.

P28b

1 C4-activating PlOO

d

component

i

70 LD-ctmia

* 29 tpchai.

FIGURE 8. Polypeptides found in RaRF complex. Shaded box, collagen-like domain of P28a and P28b; triangle, DFPbinding site in P100.

the low m.w. components of the RaRF complex. The following results in the present study clearly indicate that a serine-type proteinase, capable of activating C4 and C2, is present in the mouse RaRF. DFP inhibited the C4-activating ability of RaRF on ELPS (Fig. 4). A DFP-binding proteinwith an apparent m.w. of 100,000 wasfound in the RaRF complex (Fig. 5). and it generated a 29-kDa polypeptide upon reduction. A DFP-binding protein of this size was recovered from a C4-consuming fraction prepared by gel-permeation chromatography of EDTA-treated RaRF (Fig. 7). The cleaved products of C4 shown in Figure 2 indicate that there exists, in the RaRF complex, a proteinase thatisvery similar to CIS in terms of the specificity for C4 as substrate and the site of cleavage of

577

Journal of Immunology

Sequence analysis indicates that the domain construction the substrate. The activated C4 bound to ELPS-RaRF. In of these 28-kDa polypeptides is similar to that of the Clq the previous report, we speculated that the classical C3 convertase, C 4 b k is generated on the ELPS-RaRF bechains and mannan-binding proteins (21). Like these cause the activities of c, and c; remain after washing the polypeptides (22, 23), both P28a and P28b contain a colcells. The Present results described above Support this lagen-like sequence located near the amino-terminus speculation. (Fig. 8). Therefore, it is thought that the binding component of RaRF corresponds to rat MBP. It has been In contrast to the function, the molecular size of the 100-kDa polypeptide in RaRF is distinct from that of Cls. reported that rat and human MBP activates C l r and C l s The electrophoretic mobilities of the DFP-binding proteins and thereby activates C4 (24, 25). However, the binding of RaRF were apparently different from thoseof mouse C 1 component of RaRF does not activate C4 when it is sep(Figs. 5 and 7). Therefore, the C4-activating proteinase in arated from the CCactivating component (8). Therefore, it RaRF is distinct from C l r and CIS. The molecular mass of the major DFp-binding protein is strongly suggested that the function of the binding comin the C4-consuming fraction was estimated to be 100 kDa ponent of mouse RaRF complex is different from the rat not only from its mobility during PAGE but also fromitsandhuman MBP. elution volume during gel-permeation chromatography. Upon reduction, the unreduced 100-kDa molecule generAcknowledgments ated 29-kDa DFP-binding and 70-kDa nonbinding chain. Therefore, the C4-activating proteinase with an apparent m.w. of 100,000 is formed by covalent binding via disulfide bonds of two chains with apparent m.w. of 29,000 and 70,000, and that the 29-kDa chain includes the active site of a proteinase (Fig. 8). It hasbeen demonstrated by a serologic methodthatthe 70-kDa chain in reduced RaRF is essential for the C-dependent bactericidal reaction of RaRF (9). This observation underlines the necessity for the 70-kDa chain, as well as of 29-kDa DFPbinding chain, of the 100-kDa polypeptide. The 70-kDa chain is probably the recognition domain (19) of the enzyme, including a site for binding to P28a or P28b polypeptide. The DFP-binding proteins demonstrated by the SDSPAGEof RaRF (Figs. 5 and 7) appears tobe the C4activating proteinase that is sensitive to DFP. In the RaRF complex, there may be more than one kind of proteinase, because C1 contains two serine proteinases, C l r and CIS. The Clt, which has been activated by the conformation change of Clq, activates Cls. However, both C4 and C2 are activated by a single enzyme, C l s inC1 (18, 20). Assuming an analogy with C1, we propose that a single proteinase in RaRF is able to cleave both C components. This hypothesis is supported by the fact that both C4- and C2-consuming activities are found in the same fraction in gel-permeation chromatography. However, yields of activity to consume each of C4 and C2 after fractionation by gel-permeation chromatography were different. It remains necessary to prove that a highly purified PlOO preparation cleaves C4 and C2. The C1 component of C is composed of an antibodyrecognizing subcomponent, Clq, and the proteinase subcomponents C 1r and C 1s. Two kinds of 28-kDa polypeptide in the RaRF complex, P28a and P28b, are similar to the A, B, and C chains of Clq and to the mannan-binding proteins of rats in terms of amino acid composition (8).

We thank Hidechika his

Okada, Nagoya City University, Nagoya, Japan for and suggestions,

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