Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b

Molecular Immunology, Vol. 32, No. 5, pp. 355-360, 1995

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HUMAN FACTOR H AND C4b-BINDING PROTEIN SERVE AS FACTOR I-COFACTORS BOTH ENCOMPASSING INACTIVATION OF C3b AND C4b TSUKASA SEYA,* KIMIYO NAKAMURA, TAKAHISA MASAKI, CHIKAKO ICHIHARA-ITOH, MISAKO MATSUMOTO and SHIGEHARU NAGASAWAS Department of Immunology, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537 , Japan; and IDepartment of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060 , Japan (First received 22 July 1994; accepted in revisedform 24 October 1994)

Abstract-Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and factor I can cleave methylamine-treated C4 (C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase. Key words: complement

regulatory proteins in blood plasma, pH optima, proteolytic enzyme.

INTRODUCTION Activation of the complement system proceeds principally on foreign cell membranes. Antibody-Cl complex and C3/C5 convertases are formed on membrane molecules competent of accepting C3b or C4b (Law and Reid, 1988). In turn, they activate C4 and C3, respectively, and spread over the convertase sites on membranes, resulting in immune cytolysis (Law and Reid, 1988). During the stage of C4 and C3 activation, large amounts of C4b and C3b fail to anchor onto the membranes and come off into the fluid-phase (Law and Reid, 1988). They are still capable of the formation of C3 convertases (C4b2a or C3bBb) in the fluid-phase. Human plasma provides two inhibitors for C3 convertases, namely factor H (Whaley and Ruddy, 1976; Pangbum et al., 1977) and C4b-binding protein (C4bp) (Nagasawa and Stroud, 1977; Fujita and Nussenzweig, 1979), which block convertase activity by two modes, accelerating the decay of the convertase complex or serving as a cofactor for factor I which proteolytically inactivates C3b/C4b (Crossley and Porter, 1980). Thus, they prohibit fluid-phase consumption of C factors secondary to the activation of C3 and C4. C4bp accelerates the decay of the classical pathway C3 convertase, C4b2a, and cleaves, together with factor I, *Author to whom correspondence should be addressed. C, complement; C3ma, methylaminetreated C3; C4ma, methylamine-treated C4; C4bp, C4b-binding protein; DACM, N(dimethylamino-4-methylcoumarinyl)maleimide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Abbreviations:

both C3b and C4b into inactive forms (Nagasawa and Stroud, 1977; Fujita and Nussenzweig, 1979). Its major role is to block the classical pathway. Factor H on the other hand, has been functionally characterized as an alternative pathway inhibitor, since it mainly accelerates the decay of the alternative pathway C3 convertase, C3bBb (Pangburn et al., 1977; Weller et al., 1977). Their

relative potencies have been characterized (Pangbum, 1986; Seya et al., 1985). The factor I-cofactor activity of factor H, however, has not been well defined. Early studies indicated that factor H inactivated C4b as well as C3b, as a cofactor for factor I (Whaley and Ruddy, 1976; Pangburn et al., 1977). However, subsequent studies corrected this point and the current concept is that factor H does not serve as a C4b-inactivating cofactor (Gigli et al., 1978; Fujita and Nussenzweig, 1979). All of the factor I-cofactors except for factor H, i.e. C4bp, CR1 and MCP, act on both C3b and C4b. For this reason, we re-examined in the present study whether factors H and I cleave C4b to C4bi or C4c + C4d. MATERIALS Complement,

reagents

AND METHODS

and antibodies

A fluorescent SH reagent, N-(dimethylamino-4methylcoumarinyl)maleimide (DACM) was purchased from Wako Pure Chemicals, Tokyo. Iodogen (Pearse Co., Richmond, IL) was used for ‘251-labeling of proteins. Human C4bp (Nagasawa et al., 1982), factor H (Seya and Nagasawa, 1985) and factor I (Nagasawa et al., 1980) were purified from pooled plasma as described previously. Factor H was also purified by other methods described by 355

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Nagaki er al. (1978) and an immuno-affinity method, and used as additional lots of the preparation. Human C3 and C4 (Nagasawa and Stroud, 1980) were purified by the reported methods, and treated with 200 mM methylamine, yielding C3ma and C4ma, respectively (Seya and Nagasawa, 1985; Ichihara et al., 1981). In some assays for determination of cofactor activity, DACMlabeled C4ma and C4b were utilized. The latter was prepared by the treatment of C4 with Cls (Matsumoto and Nagaki, 1986) and then DACM (Seya et al., 1986a) and used as a substrate instead of C4ma (Ichihara et al., 1981). Common C4A and C4B isotypes were purified from sera of two individuals with C4A3.2 and C4B2.1, respectively (Masaki et al., 1991) and also converted to C4ma (Masaki et al., 1992). These were labeled with ‘25I by the Iodogen method (Masaki et al., 1991). Specific activities were l-2 x 10’ cpm/pg in both C3ma and C4ma. A fluorescent SH reagent, DACM, was used as a tracer for the C3d and C4d portions (Seya and Nagasawa, 1985; Ichihara et al., 1981). These proteins were all stored in aliquots at -70°C. Rabbit polyclonal antibodies against factor H, C4bp, CR1 and MCP were raised in our laboratory as reported previously (Seya et al., 1990u). A goat polyclonal antibody against factor I was purchased from Kent Lab (Redmond, WA).

Sodium dodecyl sulfate-polyucrylumide gel electrophoresis (SDS-PAGE) SDS-PAGE was performed by the method of Laemmli (1970). Samples were reduced with 5% 2-mercaptoethanol. After electrophoresis, gels were dried and analysed by autoradiography using XAR-5 film (Kodak, Rochester, NY) and Cronex intensification screens (DuPont, Wilmington, DE). Assay for determination of factor I-cofactor activity In the fluid-phase assay, the fluorescence- or ‘251-labeled substrates, C4ma or C3ma (2-5 pg), were incubated at 37°C with 0.5 pg of factor I and various amounts of factor H or other cofactors, which are indicated in the figure legends. pH and ionic strength in the buffer were determined at 24°C. The doses of these reagents, times, buffer ionic strength and pH for incubation were properly determined according to the methods used in previous trials (Seya et al.. 19906; Adams et al., 1991; Maeda et al., 1993). At timed intervals, the reaction was stopped by the addition of 3 ~1 of 2-mercaptoethanol and 10 ~1 of 10% SDS, and the samples were resolved on SDS-PAGE and autoradiography. Percentage cleavage was determined with SDS-PAGE followed by analysis by fluorescence spectrophotometry (Seya and Nagasawa, 1985).

C4bp

H EO-

80

4.5

5.5

-o-

12.4

-

6.7 mS

6.5

7.5

mS

8.5

9.5

4.5

5.5

6.5

7.5

8.5

9.5

Pi-l

.d

4060- . 801

G:bpTp

g

+

to-

12.4

+-

3.2 12.4 mS mS

zo-

mS

E 0

r

4.5

5.5

I

6.5

7.5

PH

.

1

8.5

0

9.5

4.5

5.5

6.5

7.5

8.5

9.5

PH

Fig. 1. Effect of pH on the factor I-mediated cleavage of C3ma and C4ma by function of factor H or C4bp. Constant amounts of DACM-labeled C3ma (upper panels) or C4ma (lower panels) and factor I were incubated for 1-4 hr at 37°C with 3 pg of C4bp (left panels) or 3 pg of factor H (right panels). The buffer pH was adjusted by the addition of 20 mM acetate buffer (below 5.5) 20 mM phosphate buffer (6.0-7.5) or 25 mM Tris-HCl (over S.O), and the buffer conductivity was adjusted as indicated by the addition of NaCl solution. Percentage cleavage of C3ma or C4ma was determined as described by Seya and Nagasawa (1985).

Factor H is a C4b inactivator

The stroma of EAC 14b (C4b-labeled) was prepared as described previously (Mayer, 1961; Seya and Atkinson, 1989) and also used as a substrate in the presence of 0.2% NP-40 (Seya et al., 1990b). Constant amounts of factor 1 (0.5 pg) and variable amounts of cofactors were added to the substrate. The degradation profile of the C4b in the EAC14b was analysed by SDS-PAGE and autoradiography as above.

357 WA

P” Mr (x lo-3 kDa) 6.0 7.5 6.0 7.5

116 -a -C4b’

93

a

--P 66 I

RESULTS pH-dependent inactivation of C3ma and C4ma by factor I and C4bp or factor H

Two substrates, DACM-labeled C3ma and C4ma, were cleaved by factor I and cofactors in the fluid-phase, and percentage cleavage of their c( chain was determined by SDS-PAGE and spectrofluorometry. Proteolysis of C3ma and C4ma was not observed until cofactor protein, C4bp or H, was added to these substrates and protease (data not shown). The assay conditions were determined and pH optima were examined for each cofactor (Fig. 1). As reported previously (Fujita and Nussenzweig, 1979), C4bp cleaved both C3ma and C4ma, and mediated C4ma cleavage more efficiently than that of C3ma. There was a single pH optimum, pH 6.0, for C3ma cleavage, while two pH optima, pH 6.0 and pH 8.5, were observed for C4ma cleavage (Fig. 1 a,b). Factor H did not cleave C4ma under physiological conditions (Fig. Id). Under low ionic strength and acidic conditions however, it cleaved C4ma together with factor I (Fig. Id) with a pH optimum of 6.0. Factor H has been shown to serve as a cofactor for factor I-mediated cleavage of C3ma (Whaley and Ruddy, 1976). A representative pH effect on C3ma cleavage is shown in Fig. lc, which was obtained by setting the ionic strength as that under physiological conditions. If the ionic strength was set below 12 mS, the pH optimum shifted to the basic area (Fig. Ic and Seya and Nagasawa, 1985). However, regardless of ionic strength conditions, only a single pH optimum peak was obtained. C4ma is a substrate for factor I and factor H under low conductivity and acidic conditions

Since the current concept is that factor H is a cofactor for C3b but not C4b, our result shown in Fig. Id is contradictory. Methylamine-treated radiolabeled C4A isotype (C4Ama) was used as a substrate, and the cleavage profile was examined by SDS-PAGE and autoradiography (Fig. 2). Weak cofactor activity of factor H for the cleavage of C4Ama to C4d was detected under acidic pH around 6.0 and low conductivity conditions. Similar results were obtained using DACM-labeled C4b as a substrate (data not shown). C4bp expressed sufficient cofactor activity for C4ma cleavage under all pHs employed. To exclude the possibility that C4ma was cleaved by other cofactors contaminating our factor H preparation, blocking studies were performed using antibodies against known cofactors (Fig. 3). Methylamine-treated radio-

--Y Factor I

0.5

Factor H C4bp

4.0 0

0.5 4.0 0

0.5 0 1.0

0.5 0 1.0

dye front pg pg @g

Fig. 2. Generation of C4d by factor H and factor I. The buffer conductivity was adjusted to 3.2 mS/cm, and the pH was adjusted by phosphate buffer as indicated. ‘251-labeled C4Ama (5 pg) was incubated for 2 hr at 37°C with the reagents indicated, and then resolved by SDS-PAGE and autoradiography. The C4d band was confirmed using the DACM-labeled C4ma as a substrate.

labeled C4ma (prepared from pooled plasma) was used as a substrate. Under the conditions used (3.2 mS and pH 6.0, see Materials and Methods), C4ma was converted into C4c and C4d by factor I and our purified factor H even in the presence of the large amounts of polyclonal antibodies against CRl, MCP, C4bp or DAF (Fig. 3), Antibodv Mr

(x

lo-3

kDa)

200 116, ,a c C4b’ a

93-

-P

6645-

- C4d

-Y w dye front 1

23

45

67

6

Fig. 3. Blocking of C4d generation by anti-factor H or anti-factor I. The substrate, ‘*‘I-labeled C4ma, is shown in lane 1. The conditions were set as in Fig. 2 so as to generate C4d by factor I and factor H, and the incubation was performed in the presence of polyclonal antibodies against CR1 (lane 2), MCP (lane 3), C4bp (lane 4), factor H (lane 5), DAF (lane 6) and factor I (lane 8). Approximately 20 pg of the antibodies were used for this blocking study. Non-immune rabbit IgG (20 pg) was used as control (lane 7). The samples were analysed by SDS-PAGE and autoradiography.

T. SEYA et al.

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although they were capable of blocking respective cofactor activity (Seya or rll., 1990~). The 45 kDa fragment was confirmed to be C4d, since the yielded C4d showed the same electrophoretic mobility as those generated by C4bp (Fig. 2) and the fragment possessed the SH residue originating from the ruptured thioester bond into which the fluorescent SH reagent, DACM. was incorporated (Ichihara et al.. 1981). The C4d-generating activity was inhibited by polyclonal antibodies against factor H or factor I (Fig. 3). Thus, C4d was generated only by factor H and factor I (Fig 3). The results were confirmed with two other preparations of factor H purified by different procedures (data not shown).

116 93

q

45

C4d

1 30 1

Factor Handfkrtor

1 cleave both C4A ard C4Bphelzotype.s

1

Radiolabeled methylamine-treated C4B (C4Bma), in addition to C4Ama, was used as a substrate for factor I and factor H. C4Bma also underwent proteolysis into C4d (Fig. 4). Low ionic strength and acidic pH conditions were similarly critical as in the case of C4ma. The anti-factor H antibody also blocked the generation of C4d in this case. In contrast. C4bp served as a cofactor for both C4A and C4B under all pH conditions tested (Figs 2 and 4). Cleatlage hv_ fucfor. I ~mtl c.ofkct0r.s of‘ C4b membrane constituetus

bound

1

2

3

45

6

116

-a-

93

-C4b’

a-

66 -

C4d

y

30

Factor Factor Anti-H

I

0.5

0.5

0.5

Ii

0

4.0

4.0

20

0

20

0.5

C4bp

0 20

0.5

1.0 0

similar potency as 1 /lg of C4bp in the fluid-phase, cleaved C4b to C4c + C4d. A high dose of C4bp on the other hand, yielded the factor I-dependent C4b cleavage products similar to MCP and CR1 (data not shown). Even under conditions in which fluid-phase C4ma was cleaved by factor H and factor I, EAC4b was not degraded by factor H and factor I (Fig. 5). DISCUSSION In the present study, we demonstrated that human factor H serves as a C4ma-inactivating cofactor for factor I in the fluid-phase. The conditions are a critical factor for this activity: low ionic strength and acidic pH (around 6.0) are essentially required for C4ma inactivation. The functional properties of factor H capable of interacting with both C4b and C3b highlights the general nature of the C-regulatory proteins, the genes of which are clustered on the long arm of chromosome 1 (reviewed by Hourcade et al., 1989). Firstly, all the known cofactors are capable of interacting with both C4b and C3b, and factor H is not an exception (Table 1). Thus, they all must provide domains for both C4b and C3b. This issue reinforces the

C4B klla)

Fig. 5. Cleavage of EAC14b by factor I andcofactors. The buffer used in this assay was 20 mM phosphate buffer containing 0.2% NP-40. pH 6.0. The solubilizer was required to express cofactor activity of MCP and CR1 (Seya and Atkinson. 1989). The solubilized substrate (lane 1) was incubated for 2 hr at 37~C with factor I alone (lane 2), factor I plus C4bp (0.1 pg, lane 3; 1 pg, lane 4; and 2 pg, lane 5), factor I plus MCP (0.2 pg) (lane 6) factor I plus CR1 (1 pg) (lane 7), and factor I plus factor H (4 pg). The samples were analysed by SDS-PAGE and autoradiography.

lo

EAC14b was prepared using radiolabeled C4, and the supernatant of the solubilized material was used as a substrate for factor I (Seya and Atkinson, 1989). On the basis of weight, MCP and CR1 more effectively cleaved EAC4b than C4bp in the presence of a constant amount of factor I (Fig. 5). Since the x 1 fragment was barely generated by factor 1 and C4bp, C4bi was a major product for low doses of C4bp. The doses of MCP and CRl, with

Mr(xlO-3

2345678

0.5

pg

1.0

pg

20

1’9

Table 1. Functional predominance of factor I-mediated cleavage of C4b and C3b in each cofactor and their optimal pH

Fig. 4. C4Bma as a substrate for factor I and factor H. “51-labeled C4Bma is used as a substrate instead of C4Ama under the conditions used in Fig. 2. C4Bma was incubated with the reagents indicated, and after the termination of the reaction, analysed by SDS-PAGE and autoradiography. The C4d band was indicated, which was generated by factor I and factor H or C4bp and blocked with 20 [Lg of anti-H or anti-C4bp.

C4b Factor C4bp CR1 MCP

H

6.0 6.0,8.5 6.0 6.0

C3b << > = <

6.0 6.0 7.5 6.0

Factor H is a C4b inactivator

concept that the C-regulatory proteins are structurally, functionally and evolutionally related proteins. Furthermore, factor H, like other cofactor proteins, does not discriminate between C4 isotypes. Although C4B is more hemolytically active than C4A (Isenman and Young, 1984), the degradation rates of the C4 isotypes by factor I and factor H seem to be similar. Since C4bp has no decay-accelerating activity for C3bBb, as does factor H for C4b2a (Seya et al., 1985), cofactor activity which cannot discriminate C4b and C3b appears to be a more primitive function than decay-accelerating activity. Secondly, pH and conductivity induce some conformational changes in the cofactor proteins or the substrates C3b (Nilson-Ekdahl et al., 1990), and their notion may be expanded upon in C4b. This idea explains the mechanism whereby these buffer conditions modulate the potency of C4b-inactivating cofactor activity. This issue is in part consistent with a previous finding that the binding affinity of cofactors to C3b or C4b increases concomitantly with decreases in buffer ionic strength (Fisherson et al., 1987; Adams et al., 1991; Seya et al., 1990b).

Membrane-bound C4b was found to be more resistant to factor H and factor I-mediated inactivation than fluid-phase C4ma or C4b. This is reminiscent of previous findings in that (1) the other fluid-phase cofactor, C4bp, is also less effective for inactivation of membrane-bound C4b than of fluid-phase C4b (Fujita and Tamura, 1983), and (2) fluid-phase C3b is conformationally different from membrane-bound C3b (Nilsson et al., 1990, 1992; Becherer et al., 1992). These findings suggest that a certain conformational difference exists between the fluid-phase and the membrane-bound forms of C4b, as is the case for C3b. In contrast, CR1 and MCP effectively act on membrane-bound C4b as well as fluid-phase C4b (Medof et al., 1982; Seya and Atkinson, 1989). Thus, the fluid-phase cofactors may have evolved in a manner more suitable for inactivation of fluid-phase active fragments of C3 and C4. We have no evidence that the C4b inactivation by factor H is physiologically functional. Indeed, factors H and I can cleave C4b, but the optimum reaction occurs at acidic pH and low conductivity in the in vitro assays. Although the possibility cannot be excluded that natural micro-environments in the body organs (such as kidneys) create suitable conditions for factor H to control soluble or bound C4b, the regulatory potency of C4b by factor H might be negligible at least under physiological conditions. Whaley and Ruddy (1976) and Pangbum et al. (1977) initially suggested that factor H cleaves C4b together with factor I under physiological conditions. Subsequent reports however, did not support this suggestion (Gigli et al., 1978; Schreiber et al., 1978; Fujita and Nussenzweig, 1979). At that time, the properties of C4bp, which serves as a major C4b inactivator in human plasma, were poorly understood, and soluble forms of membrane regulatory proteins, CR1 (Yoon and Fearon, 1985) and MCP (Purcell et al., 1989; Hara et al., 1992) which express strong cofactor activity toward fluid-phase C3b/C4b, had

359

not been identified in plasma. In the early studies by Whaley (1976) and Pangburn (1977), these factors contaminating their factor H preparations may have facilitated factor I-mediated C4b cleavage under physiological conditions. The current concept is that C4bp (Nagasawa and Stroud, 1977) and two membrane-associated C-regulatory proteins, CR1 (Fearon, 1979; Iida and Nussenzweig, 1980) and MCP (Seya et al., 1986b), serve as cofactors for C4b inactivation as well as C3b inactivation, at least in the fluid-phase. In this regard however, the functional spectra of factor H have not been clearly determined, and this has been clarified in the present report. Hence, all known cofactors encompass both C3b and C4b inactivation by factor I (Table 1). Acknowledgements-We are grateful to Drs H. Akedo, S. Miyagawa, M. Hatanaka (Center for Adult Diseases Osaka) and Dr K. Takahashi (Hokkaido University) for their helpful discussions. This work was supported in part by grants from the Nagase Science and Technology Foundation, the Ryoichi Naito Foundation, the Mochida Memorial Foundation and the Naito Memorial Foundation.

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