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Regulatory system of guinea-pig complement C3b: Tests for compatibility of guinea-pig factors H and I with human factors
Molecular Immunology, Printed in Great Britain.
Vol.28,
No. 4/5, pp. 315-382,
OMl-ss90/91 $3.00 f0.00 Pergamon Press plc
1991
REGULATORY SYSTEM OF GUINEA-PIG COMPLEMENT C3b: TESTS FOR COMPATIBILITY OF GUINEA-PIG FACTORS H AND I WITH HUMAN FACTORS* TSUKASASEYA,~MICHIYOOKADA,KAORUHAZE@ and SHIGEHARU NAGASAWA$ Department of Immunology, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537, Japan; $Faculty of Pharmaceutical Sciences, Tokyo University, Hongo, Bunkyo-ku, Tokyo 113, Japan; and $Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan (First received 2 April 1990; accepted in revised form 3 August 1990)
Abstract-Two proteins that are involved in cleavage of methylamine-treated C3 of guinea-pig origin (C3~A)gp) have been isolated from guinea-pig serum. One of them functioned as a cofactor of human factor I (ihu) for cleavage of C3(MA)~ and its molecular size was 150 kDa. The other was functionally pure and able to cleave C3(MA)~ together with human factor H (Hhu). They appear to be analogous to human factors H and I in the guinea-pig and will be referred to as Hgp and Igp. Methylamine-treated human C3 [C3(MA)hu] was not a compatible substrate for Hgp or Igp: little cleavage of C3(MA)hu was observed if human factor H (Hhu) or I was substituted with the guinea-pig counterpart. C3(MA)gp, on the other hand, served as a substrate, though less efficiently, for Hhu and Ihu. Human C4b-binding protein (C4bp) and membrane cofactor protein (MCP) as well as Hhu could participate in cleavage of C3(MA)gp by Igp or Ihu. In these assays, C3(MA)gp was degraded again less efficiently than C3(MA)hu. Interestingly, human C3b/C4b receptor (CRl) mediated factor I-dependent cleavage of C3(MA)hu and C3(MA)gp to a similar extent regardless the sources of factor I. These results suggest that factor I-dependent C3b regulatory system is species-specific except in the case of CRl, which may function as a cofactor irrespective of species.
INTRODUCTION Guinea-pigs have been shown to possess a complement system analogous to that of humans (Linscott and Nishioka, 1963; Nelson et af., 1966). Guinea-pig C3 (C3gp)3 has a latent internal thioester which acts as a binding site to targets (Thomas and Tack, 1983), similar to human C3 (C3hu) (Law and Levine, 1977). C3gp is activated both via the classical and the alternative pathways, and the deposition of activated C3 occurs on targets. This plays a key role in further
*This work was supported in parts by grants from the Mochida Memorial Foundation, the Naito Memorial Foundation, the Sagawa Cancer Research Foundation, and the Cell Science Research Foundation. TAuthor to whom correspondence should be addressed: Dr T. Seya, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537, Japan. Abbreviations: C3(MA), methylamine-treated C3 which is functionally analogous to C3b [(hu), human origin; (gp), guinea-pig origin]; C3fMA)i, a C3bi analogue which is produced from C3(MA) by treatment with factors H and I; C4bp, C4b-binding protein; CRl, C3b/C4b receptor (CD3.5); CR2, C3d/Epstein_Barr virus receptor (CD21); DACM, N-(dimethylamino-4-methylcoumarinyl)maleimide, DACM-labeled C3(MA) or C3(MA)i is designated as f-C3(MA) or f-C3(MA)i; DAF, decay-accelerating factor (CD55); EACA, Eaminocaproic acid; HPLC, high-performance liquid chromatography; MCP, membrane cofactor protein (CD46); SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.
activation and amplification of the guinea-pig complement pathway (Nelson et al., 1966; Tamura and Nelson, 1967). If the deposited C3b remains intact, it takes part in formation of the C3jCS convertases leading to activation of the late complement components to allow the formation of the membrane attack complex (Fearon and Austen, 1977). Human complement system provides two plasma complement regulatory proteins factor H (Whaley and Ruddy, 1976; Nagaki et al., 1978) and C4bbinding protein (C4bp) (Nagasawa et al., 1976; Scharfstein et al., 1978), which accelerate the decay of the convertases and act as cofactors for plasma protease factor I to limit complement activation in body fluid (Fujita and Nussenzweig, 1979). Recent studies have highlighted the presence of several cell-associated regulatory factors (Fearon, 1979; Nicholson-Weller et al., 1982; Seya et al., 1986), which may be engaged in restricting activation of the autologous complement on host cells. Mechanisms by which host cells are protected from autologous complement attack, may in part be explained by these cell-associated complement inhibitors. Although in guinea-pigs, C4bp (Burge et al., 1981) and decay-accelerating factor (DAF) (NicholsonWeller et al., 1982) have been purified, studies on guinea-pig regulatory proteins have been poorly performed. Thus, it was unclear whether or not regulatory proteins other than C4bp and DAF are present in guinea-pig blood. Further, the CompatibiIity of
375
guinea-pig regulatory proteins with human C3b has not been investigated. For this investigation, we have purified guinea-pig factor H (Hgp), and identified guinea-pig factor I (Igp). A C3b-like form of C3hu was found to be incompatible with Hgp and Igp, while that of C3gp was compatible, though poorly, with Hhu or Ihu. Human C3b/C4b receptor (CRl) served best as cofactor for Igp, while human H, C4bp, and MCP served as poor cofactors. We therefore suggest that factor I-mediated C3b regulation is species-specific, except in the case of CRI. MATERIALS AND METHODS
Vernon complement Frofei~~, ~~fi~odie~ and reagents
Human complement components, C3 (C3hu) (Nagasawa and Stroud, 1977) factor H (Hhu) (Seya and Nagasawa, 1985), factor I (Ihu) (Nagasawa et al., 1980), C4bp (Nagasawa et ul., 1982) CR1 (Seya et al., 1985), and MCP (Seya et al., 1986) were purified as previously described. Rabbit antibody directed against Hhu was prepared previously (Seya and Nagasawa, 1985). A fluorescent SH reagent, DACM, was purchased from Wako Pure Chemical Co. Japan. The concns of the purified proteins were determined by measuring the optical density at 280 nm, assuming that E,,, “,,,, cm of 0.1% solution = 1, except for those of membrane proteins and Igp, which were estimated by the method of Kumar et al. (1985) and silver-stained SDS-PAGE, respectively. Guinea-pig complement proteins and rtntibodies
Guinea-pig sera were purchased from Nippon Biosupp. Center, Japan. Guinea-pig C3 (C3gp) was purified by the modification of the method described by Nagasawa and Stroud (1977), and Thomas and Tack (1983). Guinea-pig factor H (Hgp) was purified as a by-product of C3gp. The methods for puriftcation were as follows. The starting material (100 ml of guinea-pig serum) was treated with 1 mM of phenylmethylsulfonyl fluoride (PMSF), dialyzed against 20 mM phosphate buffer (PB)/20 mM NaCl/lO mM EDTA, pH 8.0, and then the precipitate was removed by centrifugation. The supernatant was applied to: (1) Q-Sepharose (2.5 x 50 cm, Pharmacia Co., Sweden). (a) Equilibrated (Eq.) buffer: 20mM PBjlOmM EDTA, pH 8.0. Wash buffer: the same as Eq. buffer. (b) Elute with a linear NaCl(20-400 mM) gradient in Eq. buffer and fractions having Igp activity, Ihu-cofactor activity, and A600nm> 0.01 (a marker for ceruloplasmin, see Thomas and Tack, 1983) were pooled separately as Igp. Hgp, and C3gp fractions, respectively. The Hgp (C3gp) fractions were dialyzed against 20 mM PBjlOO mM Glycine/ZO mM NaCl/20 mM EACA/lO mM EDTA, pH 6.8.
(2) Heparin-Sepharose (50 ml, Pharmacia). (a) Eq. buffer: the same as the above diffusate. Wash buffer: the same as Eq. buffer. (b) Elute with a linear NaCl(2&430 mM) gradient in Eq. buffer, and Hgp (C3gp) fractions (estimated by SDS-PAGE criteria) were pooled and dialyzed against 20 mM PB/20 mM NaCi, pH 7.5. (3) Mono Q FPLC column (Pha~a~ia). (a) Eq. buffer: the same as the diffusate. Wash buffer: the same as Eq. buffer. (b) Elute with a linear gradient of 20-500 mM NaCl in Eq. buffer; flow rate I ml/min; 1 ml fractions collected; required 60min to complete the gradient. The elution profile was monitored by LCC-500 controller (Fig. 1). Hgp was separated completely from C3gp in this step. Functionally pure factor I of guinea-pig (lgp) was prepared by a 4-step column procedure. Fractions from Q-Sepharose (see the above section) containing factor I activity, which had been collected and dialyzed against IO mM PBj20 mM t -aminocaproic acid/l 0 mM EDTA, pH 7.0, were applied to: (I) AffigeI-Blue Sepharose (200 ml, BioRad, Richmond, CA). (a) Wash with 0.5 M NaSCN and equilibrated with 20 mM PB, pH 7.0. Wash buffer: 20 mM PBi20 mM NaCI, pH 7.0. (b) Elute with a linear gradient of 20-1000mM NaCl in Eq. buffer, pH 7.0. Fractions having Igp activity collected and dialyzed against IO mM PBj20 mM EACA/lO mM EDTA. (2) CM-Sephadex C-50 (100 ml, Pharmacia). (a) Eq. buffer: 10mM MPB/lO mM EDTA, pH 6.0. Wash buffer: the same as above. (b) Elute with a linear gradient of (t-500 mM NaCl in Eq. buffer (Fig. 2). Fractions having Igp activity were pooled and dialyzed against 10 mM PB, pH 7.5. (3) Mono Q FPLC column. (a) Eq. buffer: 10 mM PB, pH 7.5. Washing buffer: the same as above. (b) Elute with a gradient of O-500 mM NaCl in Eq. buffer. The gradient was controlled as described in the above section. All columns, except for the FPLC column which was handled at room temp, were operated at 4’C. Conductivity and pH of the buffers were determined at room temp. Column running speed was controlled by a pump to be -1 ml/min. Rabbit antibody against Hgp was prepared by the reported method (Seya et al., 1987). Immunodiffusion method was used to test immunological cross-reactivity. SDS-PAGE SDS-PAGE was performed by the method of Laemmli (1970). Whenever the fluorescent intensity was to be measured, gels were cut into strips and fixed with methanol/acetate solution. Otherwise, gels were
Guinea-pig
complement
factors
H and I
Fig. 1. Elution profile of Hgp from Mono Q FPLC column. Eluate from heparin-Sepharose having Ihu-cofactor activity for cleavage of f-C3(MA)gp was applied to Mono Q column using FPLC system. Although Hgp was contaminated by trace C3gp, they were separated completely by this step. Hgp was eluted in fractions 20-23 as a sharp protein peak (280 nm absorbance) and isolated by this step. Factor I-cofactor activity of each fraction was also estimated using f-C3(MA)gp and Ihu as described (Seya and Nagasawa, 1982). Briefly, 10 pg of C3fMA)gp and 0.5 pg of Ihu were incubated with 50 ~1 of each fraction of 5 hr at 37-C. The samples were subjected to SDS-PAGE under reducing conditions. Inset is an Commassie blue-stained electrophoretogram showing that the protein peaks other than that of Hgp possessed no cofactor activity. In this figure, the degree of f-C3(MA)i generation does not always reflect the Hgp level, probably suggesting insufficient compatibility of Ihu with the guinea-pig components. C3 fragments were identified according to Ross et al. (1982). Arrows indicate protein bands contained in Ihu. Detailed conditions for elution of Hgp were described in Mareriuls and Methods.
80 40 60 FRACTION f 15 ml/ tubal Fig. 2. Elution profile of Igp from CM-Sephadex C-50. Affigel blue fractions having Igp activity (which was determined by the degree of f-C3(MA)gp cleavage) were pooled and applied to CM-Sephadex. Aliquots of the eluted fractions indicated were incubated with Hhu and f-C3(MA)gp as in Fig. 1, and cleavage of f-C3(MA)gp (Igp activity) was analyzed by SDS-PAGE under reducing conditions. Gels were stained with Commassie blue to detect any contaminants in Igp fractions.
377
378
T.
SEYA et
stained with Coomassie blue R250 or silver reagents (BioRad, Richmond, Ca).
al. RESULTS
Purification of Hgp and Igp
Assay for factor I-dependent cofactor activity Assay for factor I-cofactor activity was done using the fluorescent-labeled substrates. Methylaminetreated DACM-labeled human C3, f-C3(MA)hu, and guinea-pig C3, f-C3(MA)gp, were prepared as described (Seya and Nagasawa, 1982). These were used as the substrates for factor I and its cofactors. Cofactor assay was performed in phosphatebuffer, pH 7.4. In a typical experiment, 10 pg of the fluorescent-labeled substrate, f-C3(MA)hu or f-C3(MA)gp was incubated with 0.5 pg of factor I and one of the various cofactors for 60 min at 37°C. Concentration of the cofactors varied depending on the source. Typically, 1.0 pg of Hhu, 1.3 pg of CRl, 18 pg of C4bp, 0.025 pg of MCP and 1.2 pg of Hgp was usually employed to cleave autologous f-C3(MA) to a similar extent. Reactions were stopped by the addition of 10 ~1 of 10% SDS and 3 ~1 of 14 M 2-mercaptoethanol. The mixtures were run on SDS-PAGE. Conversion of f-C3(MA) to f-C3(MA)i was monitored by ultraviolet illumination (360 nm) and by gel scanning spectrolluorometer (Hitachi F-2000) (Seya and Nagasawa, 1982). The degree of f-C3(MA) degradation was obtained as in the case of the C3b degradation (Seya and Nagasawa, 1982, 1985).
Hgp was obtained by 3-step column procedures as a by-product of C3gp. Commercially-available guinea-pig sera was the starting material, and Hgp could be successfully monitored by its cofactor activity during column chromatography since, as mentioned later, Hgp has an ability to serve as a cofactor for human factor I. Q-sepharose was employed as the first column, from which Hgp and C3gp were eluted around 10-16 mS. Like Hhu, Hgp bound efficiently to heparin-Sepharose and by this affinity column was separable mostly from C3gp (data not shown). Fractions containing Hgp from heparin-Sepharose were pooled, dialyzed, and then applied to Mono Q column. Hgp was eluted with a linear salt gradient; the main peak of activity being around 18mS (Fig. 1). C3gp was also purified with heparin-Sepharose and mono Q column from the eluate of Q-Sepharose (data not shown). Igp was partially purified by 4 step column procedures. Aliquots of fractions were incubated with Hhu and f-C3(MA)gp and Igp activity (generation of a C3bi analogue, f-C3(MA)i) was monitored by SDS-PAGE. Elution profile of Igp from the third column, CM-Sephadex, was shown in Fig. 2. The crude Igp fractions were further subjected to Mono Q column, and, in some cases, further purification chromatography on C3(MA)gp-coupled Sepharose was performed.
c3h c3gpM,(x,()-3)
DQDIlrrl-
-
zoo-
-
116 93-
Hh HQP
Fig. 3. SDS-PAGE analysis of Hgp and C3gp in comparison with the human corresponding factors. Left panel: long of C3hu (indicated as C3”, lane 1) and C3gp (Iane 2) were analyzed by SDS-PAGE (8% gel) under reducing conditions. Open arrow head, CIchain of C3gp; closed arrow head, b chain of C3gp; molecular weight markers are supplied by BioRad Labs. Right panel: 2 pg of Hhu (indicated as Hh, lane 3) and Hgp (lane 4) were applied to SDSPAGE (8% gel) under reducing conditions. Open arrow head, Hgp; a little smaller than Hhu in molecular size. A faint band observed in the Hgp preparation at the position of 116 kDa marker, is a contaminant, since it was not recognized by antibody to Hgp by immunoblotting (not shown).
379
Guinea-pig complement factors H and I Purified Hgp gave a single band with 150 kDa and migrated slightly faster than Hhu on SDSPAGE (Fig. 3). C3gp prepared for a substrate of Igp, was also compared on SDS-PAGE with C3hu (Fig. 3). The p chain of C3gp was significantly smaller than that of C3hu, consistent with the previous report (Thomas and Tack, 1983). The Igp fraction eluted from C3gpSepharose gave two major bands with 90 and 30 kDa under nonreducing conditions on silver-stained SDS-PAGE (not shown). The 90 kDa protein could be separated from the 30 kDa protein by a molecular sieve HPLC column, TSK G3000sw (Fig. 4), and only the fractions around the 97 kDa marker expressed Igp activity (Fig. 4). The results suggested that the preparation, although still contaminated by some proteins, contained a protein with factor I activity and with molecular sizes similar to those of Ihu (Fig. 4). Rabbit polyclonal antibody to Hhu did not recognize Hgp. Likewise, antibody to Hgp did not interact with Hhu (data not shown). Functional
properties
of Hgp and Igp
In this and the following experiments, approximate concns of Igp were determined with the use of various amounts of Ihu as a standard, assuming that the 90 kDa-band was Igp. Igp and Hgp efficiently cleaved a substrate, f-C3(MA)gp. The final product was composed of an intact /l chain and two c( chain fragments (Figs 1,2), which is similar to human C3bi. However, unlike human C3bi which consists of two a2 fragments with 43 and 46 kDa (Ross et al., 1982), guinea-pig C3(MA)i had a single a2 fragment of 40 kDa. The C3f fragment (Harrison et al., 1988) may not be released or may be far smaller than that of
humans, even if it were generated, during conversion of C3(MA)gp to C3(MA)i. Using f-C3(MA)gp, we tried to determine the activity of factor H or I (Fig. 5a). Igp (0.5 pg) and 1.2pg of Hgp converted 75% of f-C3(MA)gp into f-C3(MA)i under the conditions described in Materials and Methods. Assuming that this capacity of Hgp and Igp for cleavage of f-C3(MA)gp was lOO%, replacement of Hgp with Hhu reduced the activity to u 30% (Table la). Likewise, replacement of Igp with Ihu reduced the activity to -20%. Nevertheless, f-C3(MA)gp served as a substrate even if Hhu and Ihu were used instead of Hgp and Igp, 16% of f-C3(MA)gp still being degraded by Hhu and Ihu (Table la). This point was further confirmed by kinetic analysis (Fig. 5b). f-C3(MA)hu was next employed as a substrate and similar cross-reactive test was designed. Unexpectedly, Igp and Hgp had no or little ability to cleave f-C3(MA)hu (Table lb). Although Ihu and Hhu efficiently degraded f-C3(MA)hu, the activity was almost completely lost if either one was replaced by the guinea-pig counterparts (Table 1b).
Computability with Igp for
of various cleavage
cofactors
of human
The concns of various human factor I-cofactors were adjusted so as to cleave approximately 75% of f-C3(MA)hu in the presence of 0.5pg of Ihu (Table 2). These functionally-equivalent amounts of cofactors were incubated with 0.5 pg of Igp or Ihu and f-C3(MA)gp. Hhu, C4bp and MCP markedly reduced their cofactor activities for cleavage of f-C3(MA)gp regardless of the sources of factor I. The reduction was the greatest for MCP, about 85% of
0 0.04 1
Fraction
origin
of C3(MA)gp
(1 ml/tube)
Fig. 4. Elution profile of Igp activity from a molecular sieve HPLC column. The Igp preparation (see Materials and Methods) was concentrated with Mizubutorikun (Atto Co. Janan) and subiected to a TSK G3000sw HPLC column using Pharrnacia FPLC system. Fifty microliters of each fraction was incubated with f-C3(MA)gp and Hgp, and % f-C3(MA)gp cleaved was evaluated with SDS-PAGE and spectrofluorometer as described in Materials and Methoak. Human serum and BioRad molecular weight marker were used for calibration of the column (arrow 1, initial point of serum proteins; arrow 2, myosin of 200 kDa; arrow 3, phosphorylase b of 97 kDa; arrow 4, bovine serum albumin of 66 kDa; arrow 5, ovalbumin of 43 kDa; arrow 6, end point of serum proteins).
T. SEYA el al
380
c3GP ,+,GP
Ihut tihu
iw
0 Gel
15
30
60
120
Time (min)
length
Fig. 5(b).
Fig. 5(a).
Fig. 5. Fluorometric assay for determination of Igp and its cofactor activity. (a) f-C3(MA)gp (panel A) and that treated with Hgp and Igp (panel B) were analyzed by a Hitachi spectrofluoromcter. Native C3gp
could not be labeled with DACM (not shown), and the cleavage profile of f-C3(MA)gp is essentially identical to f-C3(MA)hu, suggesting that DACM is incorporated into the SH group originated from the thioester bond characteristic of C3 (Seya ei al,, 1990). Percentage cleavage of f-C3(MA)gp was calculated by the formula previously described (Seya and Nagasawa, 1982). (b) A sample containing C3(MA)gp (50 IL&, Igp (2.5pg), and Hgp (6.Opg), and that containing C3(MA)~p (50{(g). fhu (2.5 pg). and Hhu (j.O/tg), were incubated at 37.C. These amounts of factors I and H cleaved homologous C3(MA) to a similar extent. At timed intervals, aliquots were withdrawn, mixed with 10 ~1 of 10% SDS and 3 ~1 of 2-mercaptoethanol. and then analyzed by SDS-PAGE. Percentage cleavage of a chain of C3(MA) was calculated as in the above section.
the activity being abolished. In contrast, human CR1 retained its full activity with either factor I. DISCUSSfON
The purpose of this study was to define speciesspecificity of factor I-dependent C3b-inactivation system. We have prepared human complement proteins associated with inactivation of human C3b (Nagasawa et nl., 1982; Seya and Nagasawa, 1985; Seya ef a!., 1985; Seya ef al., 1986; Seya et al., 1987). For this study, guinea-pig proteins engaged in fIuid-phase regulation for the activated C3 [C3b, C3(MA)] were additionally prepared. Guinea-pigs have a complement system that has been identified as a human analogue (Nelson et al., 1966). Mechanisms of activation of complement have been investigated using the guinea-pig system in traditional manners. Inability of human C2 to interact with guinea-pig C4b (Tamura, 19701, and, probably, of guinea-pig C5 to assemble with the mouse late Table
1. Compatibility
a
C3(MA)= IBP Ih”
of factors H and I with C3(MA) human and guinea-pigs
Hh”
100.0 22.1
30.7 16.9
IBP I””
‘Table 2. Compatibility C3(MA)= ~~ --~ I@ Ih”
H8P
Hh”
0.0 0.0
0.4 100.0
Functionally-equivalent factors H and I of human and guinea-pig origin were used for cleavage of C3(MA)hu and C3(MA)gp (see the text). In both panels a and b, the conditions were adjusted so as to cleave about 75% of the autologous substrates and this actwity was assumed to be 100%.
_
_ Hh” 25.5 16.1 (21.1%)
b
C3(MA)hU
HEP
between
complement components (Fukuoka et al., 1984) have been reported. in this study, we have purified a factor I-cofactor protein from guinea-pig plasma whose molecular mass was 150 kDa. We identified this as Hgp, since it was similar to Hhu in its molecular size (Whaley and Ruddy, 1976: Nagaki et al.. 1978), accelerated the decay of the C3 convertase (data not shown, Nagaki et a/., 1978. Ito and Tamura, 1983), possessed the ability to serve as a cofactor for factor I, Ihu and Igp (Pangburn et d., 1977; Nagaki et al., 1978). and cleaved C3fMA)gp to a C3bi analogue together with Igp. Furthermore, it had an ability to bind to C3(MA)hu and to cleave C3(MA)hu together with Igp. Factor I activity was also identified in fractions of guinea-pig sxa, and purified into SOO-fold specific activity. The factor I activity was eluted at the 97 kDa marker from the TSK G3000sw column. Although it of human
f&or
~~~_~ -. ..-. C4bph” 17.8 22.3 (30.1%)
I-cofactors
with C3(MA)@
..CRlh” ___~ 82.8 19.2 (111.2%)
MCPh” 8.8 Il.9 (15.7%)
;J(M@” 76.4
Factor
74.0
71.2
75.9
Hhu (l.Opg), C4bp (l8~1g)% CR1 (1.3pg3, and MCP (0.0X pg) of human origin cleaved ncariy the same amounts of C3(MA)hu together with Ihu (the lower column). Ability of these amounts of human cofactors to cleave the guinea-pig substrate (C3(MAjgp) together with Igp or Ihu was examined (upper column). [C3(MA)gp cleaved,KX(MA)hu cleaved] was indicated by percenrage in paronthek
Guinea-pig
complement
only partially purified, it was able to bind to C3gp. It cleaved C3(MA)gp together with Hhu or Hgp. These results suggest that guinea-pig plasma has a C3b regulatory system analogous to that in human. A complement regulatory system of guinea-pigs was first described by Tamura and Nelson (1967). Studies on the regulatory system were followed by several reports to identify the two functional entities related to the regulation, putative factors H and I (Okada et al., 1969; Kaido and Gigli, 1987). This report supports at molecular level the functional concept and gives evidence for the presence of factors H and I in guinea-pig serum. Hgp or Igp is shown to cleave C3(MA)gp together with the human partners, although the activity was markedly reduced compared with the homologous systems. This reduction in activity would reflect partial incompatibility lying between human components and guinea-pig ones. Interestingly, very little or no degradation was observed when the substrate was replaced by C3(MA)hu. High concns of guineapig serum, however, can degrade human C3b (Kaido and Gigli, 1987). What is responsible for this difference is probably the concns of Hgp and Igp and other regulatory proteins. The reason for this almostunidirectional cross-reaction is unknown. Composition of the primary structure of C3gp and C3hu will facilitate locating the difference of the cleavage sites for factor I between C3gp and C3hu. For the present, primary structure of C3hu is known (DeBruijin and Fey, 1985). In all the experiments of inactivation of C3(MA)gp by factors H and I of both human and guineapig origins, we identified only one x2 fragment of 40 kDa. However, under the same conditions, C3(MA)hu released the two a2 fragments. C3f fragment, which was recently characterized as a spasmogenic peptide (Ganu et al., 1989) may not be released during inactivation of C3b in guinea-pigs. Another observation of importance presented in this study is that human CR1 served as a cofactor for Igp, as efficiently as Ihu for cleavage of C3(MA)gp. Furthermore, C3(MA)gp was degraded as effectively as C3(MA)hu by human CR1 and Ihu, whose catalytic activity was not reduced even when the substrate was of guinea-pig origin. Therefore, no incompatibility practically underlies the interaction between human CR1 and C3(MA)gp. Of the cofactors tested, CR1 is the only factor that acted with similar efficiency on C3(MA)hu and C3(MA)gp. CR1 is free from the species restriction at least between human and guinea-pig systems. In contrast, the other three cofactors distinguished the human substrate from that of guinea-pig. MCP had lost its factor I-cofactor activity most extremely of the four cofactors tested. MCP therefore appears to be functioning under the very strict speciesspecific control. These results suggest that, except for the case of CRl, factor I-cofactor system is species-specific. was
factors
381
H and I
Complement regulatory proteins of mouse origin have been isolated. Similar species restriction of factor I-cofactor system has been shown in cleavage of human C4b by Ihu and murine C4bp (Kai et al., 1980). In addition, murine C3b appears to be more sensitive to murine factor I than to Ihu (Kinoshita and Nussenzweig, 1984; Kaido et al., 1984). Investigative reports regarding mouse CR1 are so far considerably complicated. From mouse spleen cells, Kinoshita et al. (1985) isolated a 190 kDa protein (Sl90) that binds to murine C3b. Based on its factor I-cofactor activity, they suggested that Sl90 was analogous to human CRl. On the other hand, another group offered the hypothesis that mouse p65 (Wong et al., 1985) is a human CR1 analogue and the 190 kDa protein identified by Kinoshita et al. would be analogous to human CR2 (Kurtz et al., 1989). Murine factor I regulatory system, therefore, may not be structurally or even functionally identical to the human system. Meanwhile, we have identified a CRl-like protein from guinea-pig peritoneal granulocytes (Seya et al., 1990) which may be partly consistent with C3bi-generation factors we previously reported (Seya and Nagasawa, 1983). The current idea that CR1 is an effective interspecies cofactor would be generalized if the cross-reaction tests among the proteins isolated from different species were to be similar to the results we presented in this report. Acknowledgements-We are grateful to Dr H. Akedo (The Center for Adult Diseases, Osaka) for his valuable discussion and to Dr V. Kumar (Washington University, St Louis) for the critical reading of the manuscript. REFERENCES
Burge J., Nicholson-Weller A. and Austen K. F. (1981) Isolation of C4-binding protein from guinea pig plasma and demonstration of its function as a control protein of the classical complement pathway C3 convertase. J. Immun. 126, 232. DeBruijin M. H. L. and Fey G. H. (1985) Human complement component C3: cDNA coding sequence and derived primary structure. Proc. natn. Acad. Sci. U.S.A. 82, 708. Fearon D. T. (1979) Regulation of the amplification of the C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proc. natn. Acad. Sci. U.S.A. 16, 5867. Fearon D. T. and Austen K. F. (1977) Immunochemistry of the classical and alternative pathways of complement. In Immunochemistry: An Advanced Text Book-(Edited by Glynn L. E. and Steward M. W.), p. 365. John Wiley, New York. Fujita T. and Nussenzweig V. (1979) The role of C4-binding protein and b 1H in proteolysis of C4b and C3b. J. exp. Med. 150, 267. Fukuoka Y., Seino J., Okuda T. and Tachibana T. (1984) Purification of the fourth. second and fifth components of mouse complement. Imr&nology 51, 493. Ganu V. S., Muller-Eberhard H. J. and Hugli T. E. (1989) Factor C3f is a spasmogenic fragment released from C3b by factors I and H: the heptadeca-peptide C3f was synthesized and characterized. Molec. Immun. 26, 939. Harrison R. A., Farries T. C., Northrop F. D., Lachman P. J. and Davis A. E. (1988) Structure of C3f, a small peptide specifically released during inactivation of
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the third component of complement. Complement and Inflammalion 5, 21. Ito S. and Tamura N. (1983) Inhibition of classical C5 convertase in the complement system by factor H. In?munolog~ 50, 63 I. Kai S., Fujita T., Gigli I. and Nussenzweig V. (1980) Mouse C3b/C4b inactivator: Purification and properties. J. Immun. 125, 2409. Kaido T., Fujita T., Takata Y., Natsuume-Sakai S. and Takahashi M. (1984) Simplified method for purification of mouse /I IH. Complemenf 1, 44. Kaido T. and Gigli 1. (1987) Phylogeny of C4bC3b cleaving activity: Similar fragmentationpatterns of human C4b and C3b oroduced bv lower animals. J. Immun. 139. 194. Kinoshita T.. Lavoie, S. and Nussenzweig V. (198.5) Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. II. Identification and properties of complement receptor type 1 (CR]). J. Immun. 134, 2564. Kinoshita T. and Nussenzweig V. (1984) Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. I. Isolation and characterization of factor H: the serum cofactor for the C3b/C4b inactivator. J. Immun. Meth. 71, 241. Kumar B. V., Lakshmi M. V. and Atkinson J. P. (1985) Fast and efficient methods for detection estimation of proteins. Biochem. Biophys. Res. Commun. 131, 883. Kurtz C. B., Paul M. S., Aegerter M., Weis J. J. and Weis J. H. (1989) Murine complement receptor gene family. II. Identification and characterization of the murine homolog (Cr2) to human CR2 and its molecular linkage to Crry. J. Immun. 143, 2058. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Law S. K. and Levine R. P. (1977) Interaction between the third component protein and cell surface macromolecules. Proc. mtn. Acad. Sci. U.S.A. 74, 2701. Linscott W. D. and Nishioka K. (1963) Components of guinea-pig complement. II. Separation of serum fractions essential for immunohemolysis. J. exp. Med. 118, 795. Nagaki K.. Iida K. and Inai S. (1978) Reaction mechanism of filH globulin. Inr. Archs Allergy appl. Immun. 57, 221. Nagasawa S., Ichihara C. and Stroud R. M. (1980) Cleavage of C4b by C3b inactivator; production of a nicked form of C4b, C4b’. as an intermediate cleavage product of C4b by C3b inactivator. J. Immun. 125, 578. Nagasawa S., Shiraishi S. and Stroud R. M. (1976) The molecular structure of C4 and its cleavage products. J. Immun. 116, 1743. Nagasawa S. and Stroud R. M. (1977) Mechanism of action of the C3b inactivator; requirement for a high molecular weight cofactor, C3b-C4bINA-cofactor, and production of a new C3b derivative (C3b’). Immunochemistry 14,749. Nagasawa S.. Mizuguchi ‘K., Ichihara C. and Kbyama J. (1982) Limited chymotryptic cleavage of human C4bbinding protein; isolation of a carbohydrate-containing core domain and an active fragment. J. Biochem. 92, 1329. Nelson R. A., Jr, Jensen J., Gigli I. and Tamura N. (1966) Methods for the separation, purification and measurement of nine components of hemolytic complement in guinea-pig serum. Immunochemistry 3, I1 1. Nicholson-Weller A., Burge J. and Austen K. F. (1981) Purification from guinea-pig erythrocyte stroma of a
et al
decay-accelerating factor for the classical C3 convertase, C4b, 2a. J. Immun. 127, 2035. Nicholson-Weller A., Burge J., Fearon D. T., Weller P. F. and Austen K. F. (1982) Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertase of the complement system. J. Immun. 129, 184. Okada H., Kawachi S. and Nishioka K. (1969) A new complement inhibitor in guinea pig serum. Jap. J. exp. Med. 39, 521. Pangburn M. K., Schreiber R. D. and MuIIer-Eberhard H. J. (1977) Human complement C3b inactivator: Isolation, characterization, and demonstration of an absolute requirement for the serum protein B IH for cleavage of C3b and C4b in solution. J. exp. Med. 146, 257. Ross G. D.. Lambris J. D., Cain J. A. and Newman S. L. (1982) Generation of three different fragments of bound C3 with purified factor I or serum. I. Requirement for factor H vs CR1 cofactor activity. J. Immun. 129, 2051. Scharfstein J., Ferreira A., Gigli I. and Nussenzweig V. (1978) Human CCbinding protein. I. Isolation and characterization. J. Exp. Med. 148, 207. Seya T., Farries T. C., Nickels M. and Atkinson J. P. (1987) Additional forms of human decay-accelerating factor (DAF). J. Immun. 139, 1260. Seya T., Holers V. M. and Atkinson J. P. (1985) Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CRI) and comparison with H, C4bbinding protein (C4bp), and decay-accelerating factor (DAF). J. Immvn. 135, 2661. Seya T. and Nagasawa S. (1982) A fluorescent method for determination of C3b inactivator. Clin. Chim. Acta 119, 189. Seya T. and Nagasawa S. (1983) Isolation of an iC3b forming enzyme from peritoneal polymorphonuclear leukocytes of guinea-pigs. Biochem. Biophys. Res. Commun. 117, 423. Seya T. and Nagasawa S. (1985) Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator; isolation and characterization of a biologically active fragment, C3dg. J. Biochem. 97, 373. Seya T., Okada M., Hazeki K. and Nagasawa S. (1990) Regulatory system of guinea-pig complement C3b Two factor I-cofactor proteins on guinea-pig peritoneal granulocytes. Biochem. Biophys. Res. Commun. 170, 504. Seya T., Turner J. R. and Atkinson J. P. (1986) Purification and characterization of a membrane protein (gp45-70) which is a cofactor for cleavage of C3b and C4b. J. exp. Med. 163, 837. Tamura N. (1970) An incompatibility in the reaction of the second complement of human complement with the fourth complement of guinea-pig complement. Immunology 18, 203. Tamura N. and Nelson R. A. Jr (1967) Three naturallyoccuring inhibitors of components of complement in guinea-pig and rabbit serum. J. Immun. 99, 582. Thomas M. L. and Tack B. F. (1983) Identification and alignment of a thiol ester site in the third component of guinea pig complement. Biochemistry 22, 942. Whaley K. and Ruddy S. (1976) Modulation of the alternative complement pathway by /I 1H globulin. J. exp. Med. 144, 1147. Wong W. W. and Fearon D. T. (1985) ~65, a C3b-binding protein on mouse cells that shares antigenic determinants with the human C3b receptor (CRI) and is distinct from mouse C3b receptor. J. Immun. 134, 4048.
Vol.28,
No. 4/5, pp. 315-382,
OMl-ss90/91 $3.00 f0.00 Pergamon Press plc
1991
REGULATORY SYSTEM OF GUINEA-PIG COMPLEMENT C3b: TESTS FOR COMPATIBILITY OF GUINEA-PIG FACTORS H AND I WITH HUMAN FACTORS* TSUKASASEYA,~MICHIYOOKADA,KAORUHAZE@ and SHIGEHARU NAGASAWA$ Department of Immunology, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537, Japan; $Faculty of Pharmaceutical Sciences, Tokyo University, Hongo, Bunkyo-ku, Tokyo 113, Japan; and $Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060, Japan (First received 2 April 1990; accepted in revised form 3 August 1990)
Abstract-Two proteins that are involved in cleavage of methylamine-treated C3 of guinea-pig origin (C3~A)gp) have been isolated from guinea-pig serum. One of them functioned as a cofactor of human factor I (ihu) for cleavage of C3(MA)~ and its molecular size was 150 kDa. The other was functionally pure and able to cleave C3(MA)~ together with human factor H (Hhu). They appear to be analogous to human factors H and I in the guinea-pig and will be referred to as Hgp and Igp. Methylamine-treated human C3 [C3(MA)hu] was not a compatible substrate for Hgp or Igp: little cleavage of C3(MA)hu was observed if human factor H (Hhu) or I was substituted with the guinea-pig counterpart. C3(MA)gp, on the other hand, served as a substrate, though less efficiently, for Hhu and Ihu. Human C4b-binding protein (C4bp) and membrane cofactor protein (MCP) as well as Hhu could participate in cleavage of C3(MA)gp by Igp or Ihu. In these assays, C3(MA)gp was degraded again less efficiently than C3(MA)hu. Interestingly, human C3b/C4b receptor (CRl) mediated factor I-dependent cleavage of C3(MA)hu and C3(MA)gp to a similar extent regardless the sources of factor I. These results suggest that factor I-dependent C3b regulatory system is species-specific except in the case of CRl, which may function as a cofactor irrespective of species.
INTRODUCTION Guinea-pigs have been shown to possess a complement system analogous to that of humans (Linscott and Nishioka, 1963; Nelson et af., 1966). Guinea-pig C3 (C3gp)3 has a latent internal thioester which acts as a binding site to targets (Thomas and Tack, 1983), similar to human C3 (C3hu) (Law and Levine, 1977). C3gp is activated both via the classical and the alternative pathways, and the deposition of activated C3 occurs on targets. This plays a key role in further
*This work was supported in parts by grants from the Mochida Memorial Foundation, the Naito Memorial Foundation, the Sagawa Cancer Research Foundation, and the Cell Science Research Foundation. TAuthor to whom correspondence should be addressed: Dr T. Seya, Center for Adult Diseases Osaka, Higashinari-ku, Osaka 537, Japan. Abbreviations: C3(MA), methylamine-treated C3 which is functionally analogous to C3b [(hu), human origin; (gp), guinea-pig origin]; C3fMA)i, a C3bi analogue which is produced from C3(MA) by treatment with factors H and I; C4bp, C4b-binding protein; CRl, C3b/C4b receptor (CD3.5); CR2, C3d/Epstein_Barr virus receptor (CD21); DACM, N-(dimethylamino-4-methylcoumarinyl)maleimide, DACM-labeled C3(MA) or C3(MA)i is designated as f-C3(MA) or f-C3(MA)i; DAF, decay-accelerating factor (CD55); EACA, Eaminocaproic acid; HPLC, high-performance liquid chromatography; MCP, membrane cofactor protein (CD46); SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.
activation and amplification of the guinea-pig complement pathway (Nelson et al., 1966; Tamura and Nelson, 1967). If the deposited C3b remains intact, it takes part in formation of the C3jCS convertases leading to activation of the late complement components to allow the formation of the membrane attack complex (Fearon and Austen, 1977). Human complement system provides two plasma complement regulatory proteins factor H (Whaley and Ruddy, 1976; Nagaki et al., 1978) and C4bbinding protein (C4bp) (Nagasawa et al., 1976; Scharfstein et al., 1978), which accelerate the decay of the convertases and act as cofactors for plasma protease factor I to limit complement activation in body fluid (Fujita and Nussenzweig, 1979). Recent studies have highlighted the presence of several cell-associated regulatory factors (Fearon, 1979; Nicholson-Weller et al., 1982; Seya et al., 1986), which may be engaged in restricting activation of the autologous complement on host cells. Mechanisms by which host cells are protected from autologous complement attack, may in part be explained by these cell-associated complement inhibitors. Although in guinea-pigs, C4bp (Burge et al., 1981) and decay-accelerating factor (DAF) (NicholsonWeller et al., 1982) have been purified, studies on guinea-pig regulatory proteins have been poorly performed. Thus, it was unclear whether or not regulatory proteins other than C4bp and DAF are present in guinea-pig blood. Further, the CompatibiIity of
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guinea-pig regulatory proteins with human C3b has not been investigated. For this investigation, we have purified guinea-pig factor H (Hgp), and identified guinea-pig factor I (Igp). A C3b-like form of C3hu was found to be incompatible with Hgp and Igp, while that of C3gp was compatible, though poorly, with Hhu or Ihu. Human C3b/C4b receptor (CRl) served best as cofactor for Igp, while human H, C4bp, and MCP served as poor cofactors. We therefore suggest that factor I-mediated C3b regulation is species-specific, except in the case of CRI. MATERIALS AND METHODS
Vernon complement Frofei~~, ~~fi~odie~ and reagents
Human complement components, C3 (C3hu) (Nagasawa and Stroud, 1977) factor H (Hhu) (Seya and Nagasawa, 1985), factor I (Ihu) (Nagasawa et al., 1980), C4bp (Nagasawa et ul., 1982) CR1 (Seya et al., 1985), and MCP (Seya et al., 1986) were purified as previously described. Rabbit antibody directed against Hhu was prepared previously (Seya and Nagasawa, 1985). A fluorescent SH reagent, DACM, was purchased from Wako Pure Chemical Co. Japan. The concns of the purified proteins were determined by measuring the optical density at 280 nm, assuming that E,,, “,,,, cm of 0.1% solution = 1, except for those of membrane proteins and Igp, which were estimated by the method of Kumar et al. (1985) and silver-stained SDS-PAGE, respectively. Guinea-pig complement proteins and rtntibodies
Guinea-pig sera were purchased from Nippon Biosupp. Center, Japan. Guinea-pig C3 (C3gp) was purified by the modification of the method described by Nagasawa and Stroud (1977), and Thomas and Tack (1983). Guinea-pig factor H (Hgp) was purified as a by-product of C3gp. The methods for puriftcation were as follows. The starting material (100 ml of guinea-pig serum) was treated with 1 mM of phenylmethylsulfonyl fluoride (PMSF), dialyzed against 20 mM phosphate buffer (PB)/20 mM NaCl/lO mM EDTA, pH 8.0, and then the precipitate was removed by centrifugation. The supernatant was applied to: (1) Q-Sepharose (2.5 x 50 cm, Pharmacia Co., Sweden). (a) Equilibrated (Eq.) buffer: 20mM PBjlOmM EDTA, pH 8.0. Wash buffer: the same as Eq. buffer. (b) Elute with a linear NaCl(20-400 mM) gradient in Eq. buffer and fractions having Igp activity, Ihu-cofactor activity, and A600nm> 0.01 (a marker for ceruloplasmin, see Thomas and Tack, 1983) were pooled separately as Igp. Hgp, and C3gp fractions, respectively. The Hgp (C3gp) fractions were dialyzed against 20 mM PBjlOO mM Glycine/ZO mM NaCl/20 mM EACA/lO mM EDTA, pH 6.8.
(2) Heparin-Sepharose (50 ml, Pharmacia). (a) Eq. buffer: the same as the above diffusate. Wash buffer: the same as Eq. buffer. (b) Elute with a linear NaCl(2&430 mM) gradient in Eq. buffer, and Hgp (C3gp) fractions (estimated by SDS-PAGE criteria) were pooled and dialyzed against 20 mM PB/20 mM NaCi, pH 7.5. (3) Mono Q FPLC column (Pha~a~ia). (a) Eq. buffer: the same as the diffusate. Wash buffer: the same as Eq. buffer. (b) Elute with a linear gradient of 20-500 mM NaCl in Eq. buffer; flow rate I ml/min; 1 ml fractions collected; required 60min to complete the gradient. The elution profile was monitored by LCC-500 controller (Fig. 1). Hgp was separated completely from C3gp in this step. Functionally pure factor I of guinea-pig (lgp) was prepared by a 4-step column procedure. Fractions from Q-Sepharose (see the above section) containing factor I activity, which had been collected and dialyzed against IO mM PBj20 mM t -aminocaproic acid/l 0 mM EDTA, pH 7.0, were applied to: (I) AffigeI-Blue Sepharose (200 ml, BioRad, Richmond, CA). (a) Wash with 0.5 M NaSCN and equilibrated with 20 mM PB, pH 7.0. Wash buffer: 20 mM PBi20 mM NaCI, pH 7.0. (b) Elute with a linear gradient of 20-1000mM NaCl in Eq. buffer, pH 7.0. Fractions having Igp activity collected and dialyzed against IO mM PBj20 mM EACA/lO mM EDTA. (2) CM-Sephadex C-50 (100 ml, Pharmacia). (a) Eq. buffer: 10mM MPB/lO mM EDTA, pH 6.0. Wash buffer: the same as above. (b) Elute with a linear gradient of (t-500 mM NaCl in Eq. buffer (Fig. 2). Fractions having Igp activity were pooled and dialyzed against 10 mM PB, pH 7.5. (3) Mono Q FPLC column. (a) Eq. buffer: 10 mM PB, pH 7.5. Washing buffer: the same as above. (b) Elute with a gradient of O-500 mM NaCl in Eq. buffer. The gradient was controlled as described in the above section. All columns, except for the FPLC column which was handled at room temp, were operated at 4’C. Conductivity and pH of the buffers were determined at room temp. Column running speed was controlled by a pump to be -1 ml/min. Rabbit antibody against Hgp was prepared by the reported method (Seya et al., 1987). Immunodiffusion method was used to test immunological cross-reactivity. SDS-PAGE SDS-PAGE was performed by the method of Laemmli (1970). Whenever the fluorescent intensity was to be measured, gels were cut into strips and fixed with methanol/acetate solution. Otherwise, gels were
Guinea-pig
complement
factors
H and I
Fig. 1. Elution profile of Hgp from Mono Q FPLC column. Eluate from heparin-Sepharose having Ihu-cofactor activity for cleavage of f-C3(MA)gp was applied to Mono Q column using FPLC system. Although Hgp was contaminated by trace C3gp, they were separated completely by this step. Hgp was eluted in fractions 20-23 as a sharp protein peak (280 nm absorbance) and isolated by this step. Factor I-cofactor activity of each fraction was also estimated using f-C3(MA)gp and Ihu as described (Seya and Nagasawa, 1982). Briefly, 10 pg of C3fMA)gp and 0.5 pg of Ihu were incubated with 50 ~1 of each fraction of 5 hr at 37-C. The samples were subjected to SDS-PAGE under reducing conditions. Inset is an Commassie blue-stained electrophoretogram showing that the protein peaks other than that of Hgp possessed no cofactor activity. In this figure, the degree of f-C3(MA)i generation does not always reflect the Hgp level, probably suggesting insufficient compatibility of Ihu with the guinea-pig components. C3 fragments were identified according to Ross et al. (1982). Arrows indicate protein bands contained in Ihu. Detailed conditions for elution of Hgp were described in Mareriuls and Methods.
80 40 60 FRACTION f 15 ml/ tubal Fig. 2. Elution profile of Igp from CM-Sephadex C-50. Affigel blue fractions having Igp activity (which was determined by the degree of f-C3(MA)gp cleavage) were pooled and applied to CM-Sephadex. Aliquots of the eluted fractions indicated were incubated with Hhu and f-C3(MA)gp as in Fig. 1, and cleavage of f-C3(MA)gp (Igp activity) was analyzed by SDS-PAGE under reducing conditions. Gels were stained with Commassie blue to detect any contaminants in Igp fractions.
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T.
SEYA et
stained with Coomassie blue R250 or silver reagents (BioRad, Richmond, Ca).
al. RESULTS
Purification of Hgp and Igp
Assay for factor I-dependent cofactor activity Assay for factor I-cofactor activity was done using the fluorescent-labeled substrates. Methylaminetreated DACM-labeled human C3, f-C3(MA)hu, and guinea-pig C3, f-C3(MA)gp, were prepared as described (Seya and Nagasawa, 1982). These were used as the substrates for factor I and its cofactors. Cofactor assay was performed in phosphatebuffer, pH 7.4. In a typical experiment, 10 pg of the fluorescent-labeled substrate, f-C3(MA)hu or f-C3(MA)gp was incubated with 0.5 pg of factor I and one of the various cofactors for 60 min at 37°C. Concentration of the cofactors varied depending on the source. Typically, 1.0 pg of Hhu, 1.3 pg of CRl, 18 pg of C4bp, 0.025 pg of MCP and 1.2 pg of Hgp was usually employed to cleave autologous f-C3(MA) to a similar extent. Reactions were stopped by the addition of 10 ~1 of 10% SDS and 3 ~1 of 14 M 2-mercaptoethanol. The mixtures were run on SDS-PAGE. Conversion of f-C3(MA) to f-C3(MA)i was monitored by ultraviolet illumination (360 nm) and by gel scanning spectrolluorometer (Hitachi F-2000) (Seya and Nagasawa, 1982). The degree of f-C3(MA) degradation was obtained as in the case of the C3b degradation (Seya and Nagasawa, 1982, 1985).
Hgp was obtained by 3-step column procedures as a by-product of C3gp. Commercially-available guinea-pig sera was the starting material, and Hgp could be successfully monitored by its cofactor activity during column chromatography since, as mentioned later, Hgp has an ability to serve as a cofactor for human factor I. Q-sepharose was employed as the first column, from which Hgp and C3gp were eluted around 10-16 mS. Like Hhu, Hgp bound efficiently to heparin-Sepharose and by this affinity column was separable mostly from C3gp (data not shown). Fractions containing Hgp from heparin-Sepharose were pooled, dialyzed, and then applied to Mono Q column. Hgp was eluted with a linear salt gradient; the main peak of activity being around 18mS (Fig. 1). C3gp was also purified with heparin-Sepharose and mono Q column from the eluate of Q-Sepharose (data not shown). Igp was partially purified by 4 step column procedures. Aliquots of fractions were incubated with Hhu and f-C3(MA)gp and Igp activity (generation of a C3bi analogue, f-C3(MA)i) was monitored by SDS-PAGE. Elution profile of Igp from the third column, CM-Sephadex, was shown in Fig. 2. The crude Igp fractions were further subjected to Mono Q column, and, in some cases, further purification chromatography on C3(MA)gp-coupled Sepharose was performed.
c3h c3gpM,(x,()-3)
DQDIlrrl-
-
zoo-
-
116 93-
Hh HQP
Fig. 3. SDS-PAGE analysis of Hgp and C3gp in comparison with the human corresponding factors. Left panel: long of C3hu (indicated as C3”, lane 1) and C3gp (Iane 2) were analyzed by SDS-PAGE (8% gel) under reducing conditions. Open arrow head, CIchain of C3gp; closed arrow head, b chain of C3gp; molecular weight markers are supplied by BioRad Labs. Right panel: 2 pg of Hhu (indicated as Hh, lane 3) and Hgp (lane 4) were applied to SDSPAGE (8% gel) under reducing conditions. Open arrow head, Hgp; a little smaller than Hhu in molecular size. A faint band observed in the Hgp preparation at the position of 116 kDa marker, is a contaminant, since it was not recognized by antibody to Hgp by immunoblotting (not shown).
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Guinea-pig complement factors H and I Purified Hgp gave a single band with 150 kDa and migrated slightly faster than Hhu on SDSPAGE (Fig. 3). C3gp prepared for a substrate of Igp, was also compared on SDS-PAGE with C3hu (Fig. 3). The p chain of C3gp was significantly smaller than that of C3hu, consistent with the previous report (Thomas and Tack, 1983). The Igp fraction eluted from C3gpSepharose gave two major bands with 90 and 30 kDa under nonreducing conditions on silver-stained SDS-PAGE (not shown). The 90 kDa protein could be separated from the 30 kDa protein by a molecular sieve HPLC column, TSK G3000sw (Fig. 4), and only the fractions around the 97 kDa marker expressed Igp activity (Fig. 4). The results suggested that the preparation, although still contaminated by some proteins, contained a protein with factor I activity and with molecular sizes similar to those of Ihu (Fig. 4). Rabbit polyclonal antibody to Hhu did not recognize Hgp. Likewise, antibody to Hgp did not interact with Hhu (data not shown). Functional
properties
of Hgp and Igp
In this and the following experiments, approximate concns of Igp were determined with the use of various amounts of Ihu as a standard, assuming that the 90 kDa-band was Igp. Igp and Hgp efficiently cleaved a substrate, f-C3(MA)gp. The final product was composed of an intact /l chain and two c( chain fragments (Figs 1,2), which is similar to human C3bi. However, unlike human C3bi which consists of two a2 fragments with 43 and 46 kDa (Ross et al., 1982), guinea-pig C3(MA)i had a single a2 fragment of 40 kDa. The C3f fragment (Harrison et al., 1988) may not be released or may be far smaller than that of
humans, even if it were generated, during conversion of C3(MA)gp to C3(MA)i. Using f-C3(MA)gp, we tried to determine the activity of factor H or I (Fig. 5a). Igp (0.5 pg) and 1.2pg of Hgp converted 75% of f-C3(MA)gp into f-C3(MA)i under the conditions described in Materials and Methods. Assuming that this capacity of Hgp and Igp for cleavage of f-C3(MA)gp was lOO%, replacement of Hgp with Hhu reduced the activity to u 30% (Table la). Likewise, replacement of Igp with Ihu reduced the activity to -20%. Nevertheless, f-C3(MA)gp served as a substrate even if Hhu and Ihu were used instead of Hgp and Igp, 16% of f-C3(MA)gp still being degraded by Hhu and Ihu (Table la). This point was further confirmed by kinetic analysis (Fig. 5b). f-C3(MA)hu was next employed as a substrate and similar cross-reactive test was designed. Unexpectedly, Igp and Hgp had no or little ability to cleave f-C3(MA)hu (Table lb). Although Ihu and Hhu efficiently degraded f-C3(MA)hu, the activity was almost completely lost if either one was replaced by the guinea-pig counterparts (Table 1b).
Computability with Igp for
of various cleavage
cofactors
of human
The concns of various human factor I-cofactors were adjusted so as to cleave approximately 75% of f-C3(MA)hu in the presence of 0.5pg of Ihu (Table 2). These functionally-equivalent amounts of cofactors were incubated with 0.5 pg of Igp or Ihu and f-C3(MA)gp. Hhu, C4bp and MCP markedly reduced their cofactor activities for cleavage of f-C3(MA)gp regardless of the sources of factor I. The reduction was the greatest for MCP, about 85% of
0 0.04 1
Fraction
origin
of C3(MA)gp
(1 ml/tube)
Fig. 4. Elution profile of Igp activity from a molecular sieve HPLC column. The Igp preparation (see Materials and Methods) was concentrated with Mizubutorikun (Atto Co. Janan) and subiected to a TSK G3000sw HPLC column using Pharrnacia FPLC system. Fifty microliters of each fraction was incubated with f-C3(MA)gp and Hgp, and % f-C3(MA)gp cleaved was evaluated with SDS-PAGE and spectrofluorometer as described in Materials and Methoak. Human serum and BioRad molecular weight marker were used for calibration of the column (arrow 1, initial point of serum proteins; arrow 2, myosin of 200 kDa; arrow 3, phosphorylase b of 97 kDa; arrow 4, bovine serum albumin of 66 kDa; arrow 5, ovalbumin of 43 kDa; arrow 6, end point of serum proteins).
T. SEYA el al
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c3GP ,+,GP
Ihut tihu
iw
0 Gel
15
30
60
120
Time (min)
length
Fig. 5(b).
Fig. 5(a).
Fig. 5. Fluorometric assay for determination of Igp and its cofactor activity. (a) f-C3(MA)gp (panel A) and that treated with Hgp and Igp (panel B) were analyzed by a Hitachi spectrofluoromcter. Native C3gp
could not be labeled with DACM (not shown), and the cleavage profile of f-C3(MA)gp is essentially identical to f-C3(MA)hu, suggesting that DACM is incorporated into the SH group originated from the thioester bond characteristic of C3 (Seya ei al,, 1990). Percentage cleavage of f-C3(MA)gp was calculated by the formula previously described (Seya and Nagasawa, 1982). (b) A sample containing C3(MA)gp (50 IL&, Igp (2.5pg), and Hgp (6.Opg), and that containing C3(MA)~p (50{(g). fhu (2.5 pg). and Hhu (j.O/tg), were incubated at 37.C. These amounts of factors I and H cleaved homologous C3(MA) to a similar extent. At timed intervals, aliquots were withdrawn, mixed with 10 ~1 of 10% SDS and 3 ~1 of 2-mercaptoethanol. and then analyzed by SDS-PAGE. Percentage cleavage of a chain of C3(MA) was calculated as in the above section.
the activity being abolished. In contrast, human CR1 retained its full activity with either factor I. DISCUSSfON
The purpose of this study was to define speciesspecificity of factor I-dependent C3b-inactivation system. We have prepared human complement proteins associated with inactivation of human C3b (Nagasawa et nl., 1982; Seya and Nagasawa, 1985; Seya ef a!., 1985; Seya ef al., 1986; Seya et al., 1987). For this study, guinea-pig proteins engaged in fIuid-phase regulation for the activated C3 [C3b, C3(MA)] were additionally prepared. Guinea-pigs have a complement system that has been identified as a human analogue (Nelson et al., 1966). Mechanisms of activation of complement have been investigated using the guinea-pig system in traditional manners. Inability of human C2 to interact with guinea-pig C4b (Tamura, 19701, and, probably, of guinea-pig C5 to assemble with the mouse late Table
1. Compatibility
a
C3(MA)= IBP Ih”
of factors H and I with C3(MA) human and guinea-pigs
Hh”
100.0 22.1
30.7 16.9
IBP I””
‘Table 2. Compatibility C3(MA)= ~~ --~ I@ Ih”
H8P
Hh”
0.0 0.0
0.4 100.0
Functionally-equivalent factors H and I of human and guinea-pig origin were used for cleavage of C3(MA)hu and C3(MA)gp (see the text). In both panels a and b, the conditions were adjusted so as to cleave about 75% of the autologous substrates and this actwity was assumed to be 100%.
_
_ Hh” 25.5 16.1 (21.1%)
b
C3(MA)hU
HEP
between
complement components (Fukuoka et al., 1984) have been reported. in this study, we have purified a factor I-cofactor protein from guinea-pig plasma whose molecular mass was 150 kDa. We identified this as Hgp, since it was similar to Hhu in its molecular size (Whaley and Ruddy, 1976: Nagaki et al.. 1978), accelerated the decay of the C3 convertase (data not shown, Nagaki et a/., 1978. Ito and Tamura, 1983), possessed the ability to serve as a cofactor for factor I, Ihu and Igp (Pangburn et d., 1977; Nagaki et al., 1978). and cleaved C3fMA)gp to a C3bi analogue together with Igp. Furthermore, it had an ability to bind to C3(MA)hu and to cleave C3(MA)hu together with Igp. Factor I activity was also identified in fractions of guinea-pig sxa, and purified into SOO-fold specific activity. The factor I activity was eluted at the 97 kDa marker from the TSK G3000sw column. Although it of human
f&or
~~~_~ -. ..-. C4bph” 17.8 22.3 (30.1%)
I-cofactors
with C3(MA)@
..CRlh” ___~ 82.8 19.2 (111.2%)
MCPh” 8.8 Il.9 (15.7%)
;J(M@” 76.4
Factor
74.0
71.2
75.9
Hhu (l.Opg), C4bp (l8~1g)% CR1 (1.3pg3, and MCP (0.0X pg) of human origin cleaved ncariy the same amounts of C3(MA)hu together with Ihu (the lower column). Ability of these amounts of human cofactors to cleave the guinea-pig substrate (C3(MAjgp) together with Igp or Ihu was examined (upper column). [C3(MA)gp cleaved,KX(MA)hu cleaved] was indicated by percenrage in paronthek
Guinea-pig
complement
only partially purified, it was able to bind to C3gp. It cleaved C3(MA)gp together with Hhu or Hgp. These results suggest that guinea-pig plasma has a C3b regulatory system analogous to that in human. A complement regulatory system of guinea-pigs was first described by Tamura and Nelson (1967). Studies on the regulatory system were followed by several reports to identify the two functional entities related to the regulation, putative factors H and I (Okada et al., 1969; Kaido and Gigli, 1987). This report supports at molecular level the functional concept and gives evidence for the presence of factors H and I in guinea-pig serum. Hgp or Igp is shown to cleave C3(MA)gp together with the human partners, although the activity was markedly reduced compared with the homologous systems. This reduction in activity would reflect partial incompatibility lying between human components and guinea-pig ones. Interestingly, very little or no degradation was observed when the substrate was replaced by C3(MA)hu. High concns of guineapig serum, however, can degrade human C3b (Kaido and Gigli, 1987). What is responsible for this difference is probably the concns of Hgp and Igp and other regulatory proteins. The reason for this almostunidirectional cross-reaction is unknown. Composition of the primary structure of C3gp and C3hu will facilitate locating the difference of the cleavage sites for factor I between C3gp and C3hu. For the present, primary structure of C3hu is known (DeBruijin and Fey, 1985). In all the experiments of inactivation of C3(MA)gp by factors H and I of both human and guineapig origins, we identified only one x2 fragment of 40 kDa. However, under the same conditions, C3(MA)hu released the two a2 fragments. C3f fragment, which was recently characterized as a spasmogenic peptide (Ganu et al., 1989) may not be released during inactivation of C3b in guinea-pigs. Another observation of importance presented in this study is that human CR1 served as a cofactor for Igp, as efficiently as Ihu for cleavage of C3(MA)gp. Furthermore, C3(MA)gp was degraded as effectively as C3(MA)hu by human CR1 and Ihu, whose catalytic activity was not reduced even when the substrate was of guinea-pig origin. Therefore, no incompatibility practically underlies the interaction between human CR1 and C3(MA)gp. Of the cofactors tested, CR1 is the only factor that acted with similar efficiency on C3(MA)hu and C3(MA)gp. CR1 is free from the species restriction at least between human and guinea-pig systems. In contrast, the other three cofactors distinguished the human substrate from that of guinea-pig. MCP had lost its factor I-cofactor activity most extremely of the four cofactors tested. MCP therefore appears to be functioning under the very strict speciesspecific control. These results suggest that, except for the case of CRl, factor I-cofactor system is species-specific. was
factors
381
H and I
Complement regulatory proteins of mouse origin have been isolated. Similar species restriction of factor I-cofactor system has been shown in cleavage of human C4b by Ihu and murine C4bp (Kai et al., 1980). In addition, murine C3b appears to be more sensitive to murine factor I than to Ihu (Kinoshita and Nussenzweig, 1984; Kaido et al., 1984). Investigative reports regarding mouse CR1 are so far considerably complicated. From mouse spleen cells, Kinoshita et al. (1985) isolated a 190 kDa protein (Sl90) that binds to murine C3b. Based on its factor I-cofactor activity, they suggested that Sl90 was analogous to human CRl. On the other hand, another group offered the hypothesis that mouse p65 (Wong et al., 1985) is a human CR1 analogue and the 190 kDa protein identified by Kinoshita et al. would be analogous to human CR2 (Kurtz et al., 1989). Murine factor I regulatory system, therefore, may not be structurally or even functionally identical to the human system. Meanwhile, we have identified a CRl-like protein from guinea-pig peritoneal granulocytes (Seya et al., 1990) which may be partly consistent with C3bi-generation factors we previously reported (Seya and Nagasawa, 1983). The current idea that CR1 is an effective interspecies cofactor would be generalized if the cross-reaction tests among the proteins isolated from different species were to be similar to the results we presented in this report. Acknowledgements-We are grateful to Dr H. Akedo (The Center for Adult Diseases, Osaka) for his valuable discussion and to Dr V. Kumar (Washington University, St Louis) for the critical reading of the manuscript. REFERENCES
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