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The physiological breakdown of the third component of human complement
Molecular Pergamon
Immunology* Vol 17, pp. 9-20. Press Ltd. 1980. Printed m Great tWitam.
THE PHYSIOLOGICAL BREAKDOWN OF THE THIRD COMPONENT OF HUMAN COMPLEMENT R. A. HARRISON
and P. J. LACHMANN
Department of Biochemistry, Imperial College, London SW7, and MRC Group on Mechanisms in Tumour Immunity, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England (First received 5 January
1979; in revisedfbrm
2 April 1979)
Abstract-The C3b INA*-dependent breakdown of fluid phase C3b has been shown to have an absolute requirement for a second factor. This factor is contained in catalytic amounts (with respect to C3b) of highly purified pl H. bl H alone does not cause proteolysis of C3b. In the presence of C3b INA and BlH, proteolysis of the larger? polypeptide chain (116K) of C3b occurs. Initially, a single scission gives two chains of 68K and 46K. A subsequent split of the 46K chain yields a smaller product of 43K. All of these chains remain covalently bonded to the /I chain of C3b. These initial reactions are the same whether purified components or whether low conmntrations of serum as a source of C3b INA and /31H are used. Pre-treatment of C3b, C3b INA and BIH with DFP has no effect on these events. Further protelysis of the 68K chain requires a DFP-sensitive protease and leads-to the formation of an additional high molecular weight breakdown product of C3. This has the same polypeptide chain composition as the high molecular weight breakdown product of C3 purified from aged serum, and is therefore identified as C3c.
NOMENCLATURE
Firstly, in its nascent state, it is able to bind to a wide variety of surfaces. Binding of C3b in this manner, which does not require a specific C3b receptor, confers the so-called C3 adherence reactions to the C3b binding particle. Immune (Nelson, 1953; Henson, 1969) and opsonic (Gigli & Nelson, 1968; Johnston et al., 1969) adherence are important in facilitating ingestion of this particle by phagoyctic cells that have C3b receptors on their surface (Huber et al., 1968). Secondly, the association of C3b with either the classical (Miiller-Eberhard, Dalmasso & Calcott, 1966a, b) or the alternative pathway (Vogt et al., 1978) C3 convertase gives rise to C5 convertase activity, with consequent deposition of C5b and assembly of the late acting components C6-C9 on adjacent surfaces. Thirdly, C3b itself is an intrinsic component of the alternative pathway C3 convertase (MiillerEberhard & G&e, 1972; Goodkofsky, Stewart & Lepow, 1973). This ability of C3b to amplify its own production [the so-called C3b feedback loop (Nicol & Lachmann, 1973)] clearly requires a specific homeostatic mechanism, which is provided by the C3b INA-dependent inactivation of C3b (Miiller-Eberhard & Giitze, 1972; Alper, Rosen & Lachmann, 1972). Thus, the rate at which C3b is generated by C3 convertase activity must exceed its C3b INAdependent inactivation before amplification of C3b formation is seen. Without such a system, any adventitious activation of C3 would be
Where applicable, the nomenclature agreed by the WHO Committee on Complement has been used (Bull. W.H.O. 39, 1968). INTRODUCTION
occupies a central position within the complement cascade. Following activation by a C3 convertase, it is split into two separate molecules. C3a, the smaller, is a polypeptide anaphylatoxin (da Silva, Eisele & Lepow, 1967; Bokisch, Miiller-Eberhard & Cochrane, 1969). C3b, the larger, mediates at least three major biological functions.
C3
*Abbreviationsused: C3b INA, C3b/C4b inactivator; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; CVF, cobra venom factor; PBS, phosphate buffered saline, pH 7.2; DFP, diisopropyl fluorophosphate; 2-ME, 2-mercaptoethanol; CNBr. cyanogen bromide; K. 1000 daltons. t Fragmentation of chains: C3 is composed of two polypeptide chains, previously designated a and j?, held together by disulphide bonds and non-covalent forces (Nilsson & Mapes, 1973). In the absence of an agreed nomenclature dealing with the highly complex fragmentation of the C3 a chains we have adopted a system that facilitates identification of the a-chain fragment with the trivial name for the C3 product of which it is part. Thus, the a chain of C3 contains six physiologically distinguishable portions and is written t(ddi: c3a is aa, C3d is ad, and the at portion is tentatively identified as C3e (Harrison R. A. & Lachmann P. J., in press). The presence of disulphide bridges between separate polypeptide chains is indicated by a hyphen. Native C3 is therefore c@c~B. Designation of a product as a+@+(C3c) implies only that the three chains are disulphide-bonded in an unspecified manner. 9
IO
R. A. HARRISON
and P. J. LACHMANN
expected to lead to rapid depletion of C3 and Factor B levels in serum. Genetic deficiency of C3b INA (Alper et al., 1970; Abramson et al., 1971; Thompson & Lachmaim, 1977) as well as experimental depletion of C3b INA from serum (Nicol & Lachmann, 1973) has shown this to be the case. The severe clinical consequences of C3b INA deficiency and of C3 deficiency (Alper et al., 1972) also serve to emphasize the importance of C3-mediated functions and their regulation. While the existence of C3b INA has been known for some time (Tamura & Nelson, 1967; Lachmann & Miiller-Eberhard, 1967,1968), until recently little has been known about its action on C3b at a molecular level. Early work showed that C3b INA was enzymic in nature, not being depleted during its reaction on C3b, and also that subsequent to C3b INA action on C3b, resistance to ‘trypsin-like’ enzymes was lost (Lachmann & Miiller-Eberhard, 1968). Other workers (Ruddy & Austen, 1971) suggested that C3b INA action on C3b leads directly to the production of C3c and C3d, two antigenically defined products of C3 breakdown found in aged serum. This, however, was in conflict with the known functional stages in C3b breakdown, i.e. the acquisition of conglutinin binding activity subsequent to C3b INA action but prior to the appearance of C3c (Lachmann, Elias & Moffett, 1972). Recent work has confirmed that C3c is not formed directly by C3b INA action on C3b, at least one intermediate molecular form being demonstrable (Pangburn, Schreiber & MiillerEberhard, 1977; Nagasawa & Stroud, 1977; Harrison & Lachmann, 1978). This work also showed that C3b INA by itself had no action on C3b, a co-factor being required for proteolysis to occur. We have been studying the activation and breakdown of human C3 using both purified serum components, and also serum as a source of these components, in order to elucidate the nature of possible intermediate products. In this paper the conditions under which some of these are produced, and an initial characterization of their molecular nature is given. A preliminary report of this work has been presented elsewhere (Harrison & Lachmann, 1978). MATERIALS AND METHODS
Complement
proteins
and /ll H were purified as described (Harrison & Lachmann, 1979). In some experiments, C3 that had not been passed C3
through an anti-impurity column was used. and where applicable this is indicated. Two different preparations of /?lH were used. One was about 98% pure as adjudged by SDS-PAGE, and elicited a monospecific antibody response in rabbits. The second was about 950; pure as adjudged by staining intensity on SDS-PAGE. C3c was purified from aged human serum using the method described for C3. C3b INA was purified as described by Lachmann, Nicol and Aston (1973). Factors B and D were purified essentially as described by Lachmann, Hobart and Aston (1973). Serum was freshly drawn by venepuncture from healthy donors when required, the blood being allowed to clot for 4 hr at 22°C. Preparation
of insolubilized
CVF
CVF was prepared from Naja naja kaonthia venom (Sigma) essentially’ as described by Ballow & Cochrane (1969). Purified CVF was then dialysed against 0.2 A4 citrate, pH 6.5, its concentration adjusted to 2 mg-ml- l and the CVF coupled to Sepharose CL-4B by the method of Cuatrecasas & Anfinsen (1971) under conditions described previously (Harrison & Lachmann, 1979). Preparation
of C3b
C3b was prepared from C3 by means of an insolubilized CVF-convertase essentially as described by Gitlin et al. (1975). In early experiments C3b was separated from C3a by chromatography at pH 7.2in l.OMKClandat4”Con hydroxylapatite. C3a was unadsorbed in 0.025 M phosphate, and C3b was eluted with a linear gradient from 0.025 M to 0.2 Mphosphate. C3b, at 1 mg-ml- l, was stored frozen at -20°C in 25 mM phosphate, pH 7.0, until required. Subsequent experiments showed that dissociation and removal of C3a from C3b had no effect on the breakdown of C3b and this step was discarded. DFP treatment
of protein
DFP (Sigma) was kept at -20°C as a stock solution of 1.0 M in propan-2-01 [previously dried over a molecular sieve, type 3A (BDH)]. Solutions were made 10 - * A4with DFP and held at 22°C for 1 hr and then at 2°C for 16 hr before being stored frozen at -20°C until required. Factor B activity
Factor B was measured haemolytically by the radial diffusion method of Martin et al. (1976).
The Third Component of Human Complement
11
~hrorna~o~~aphi~ media
General in~u~a~i~n and sampling procedure
DEAE-cellulose (Whatman), Sephadex and Sepharose CL-4B (Pharmacia) were used. Hydroxylapatite was manufactured as described previously (Harrison & Lachmann, 1979).
All incubations were carried out at 37°C in PBS and in capped 2 ml glass vials (Flow taboratories). At the times indicated in the text aliquots were taken into 2 ml Pyrex tubes and
gedcfb _
i
Fig. I _ The &a% of added pl H on the C3b INAdependent c&wage of C3b. The reduced proteins were rnn in SDS on an 8.5% polyacrylamide gel. Track 1: C3b INA at a loading equivalent to that in tracks 3-10. Track 2: C3b at a loading equivalent to that in tracks 3-10. Tracks 3-6: C3b (0.5 mg ml- r) + C3b INA (5 pg ml-l) after 2,20,180 mins and 24 hr incubation at 37°C. Tracks 7-10: as tracks 3-6 but with the addition of 81 H at 5 r;lgmt - *. Tracks I I: marker proteins (approximately 5 rg of each) (a) pyruvate decarboxylase, (b) catalase, (c) n-amino acid ox&se. The position of C3derived polypeptide chains is indited.
12
R. A. HARRISON
and P. J. LACHMANN
freeze-dried immediately. The freeze-dried samples were then kept at - 20°C until analysed by SDS-PAGE. If samples were required for immunochemical characterization they were frozen at -20°C immediately upon aliquoting and stored at -20°C until required.
Harrison, 1974), and ribitol dehydrogenase (Klebsiella aerogenes, 27K) (the gift of Dr. J. M. Dothie). RESULTS
(1) The requirement for a cofactor (p 1H)
SDS polyacrylamide gel electrophoresis
SDS-PAGE was carried out on polyacrylamide slabs using the buffers described by Laemmli (1970). Samples were dissolved in either 25 ~1 8.0 M urea, 1’;: mercaptoethanol, l”/, SDS (if the reduced sample was to be observed), or 25 ,uI 8.0 M urea, 19; SDS (for analysis of the nonreduced protein), and heated at 100°C for 2 min prior to loading. The final acrylamide concentration in the resolving gel varied and is indicated in the text. Gels were stained with 0.2% Coomassie Brilliant Blue G250 (Lamb). Marker proteins used were RNA polymerase from E. coli (B chain 165K, j?’ chain 155K, c1 39K), catalase (beef liver, 58K), D-amino acid oxidase (Hog kidney, 37K), myoglobin (sperm whale, 17.8K), and lysozyme (egg white, 14.3K) (all from Sigma), pyruvate decarboxylase, the E, component of the pyruvate dehydrogenase multi-enzyme complex of E. coli (103K-
lo0.000
-
40.000
-
Two parallel incubations were set up. In the first, C3b (0.5 mg-ml- ‘) was mixed with C3b INA (C3b INA:C3b 1:100 by wt). In the second, j?lH (j?lH:C3b 1:lOO by wt) was also included. Both were left for 24 hr, aliquots being taken for SDS-PAGE at 2 min, 20 min, 3 hr and 24 hr. As can be seen in Fig. 1, no cleavage of either the u (aedcfb)or fl chain of C3b occurred in the absence of PlH. In the presence of catalytic amounts of PlH, however, the 20 min and 3 hr time points showed three new bands at 68K (c&c), 46K (~fb) and 43K (&). As these appear the cledcfbchain is lost. The time course of their appearance together with their size suggests strongly that the CLedcfb chain is initially split into Cpdcand ~(fi, and that subsequent ‘trimming’ of the clfb chain produces c&, a small 3K peptide (af) being lost. During prolonged incubation (24 hr) in the presence of fil H (Fig. 1, track IO) the @dcchain is further fragmented and a succession of smaller polypeptides culminating in one at 29K are seen.
i P t B a 2 i”
61.
\
7-m \
lO.OOOl
I 02
I 04
I
I
06
0.6
Mobility Fig. 2. The molecular nature ofC3 arham-derived polypeptidechams. C3 fragmentation products were run gel and mobilities relative to bromphenol blue under reducing conditions on a 12”” SDS-polyacrylamide measured. Marker proteins used were: (1) RNA polymerase B chain; (2) RNA polymerase 8’ chain; (3) pyruvate decarboxylase; (4) catalase; (5) o-amino acid oxidase; (6) ribitol dehydrognease; (7) myoglobulin. The mobilities of C3-derived polypeptide chains are indicated by arrows. (a) a*edcth:126K; (b) aed? 1 l7K; (c) p: 75K: (d) a+ 68K: (e) a% 58K; (0 all: 46K; (g) ab: 43K; (h) a=? 39K: (i) a’ + (ad): 29K.
13
The Third Component of Human Complement
I’
aO+
C3a t C3b
C3a
I
i
a:+
t ab
C3bi
I
(C3d) (C3e) Fig. 3. Schematic representation of the breakdown of C3. (1) C3 convertase-dependent cleavage; (2) C3b INAdependent cleavage; (3) DFP-inhibitable cleavage. Arrows indicate the cleavage points. The position indicated for the a( fragment is arbitrary and it could equally well lie at the opposite end of the ab chain. It is also possible that it remains disulphide-linked to one of the chains in C3c. The interchain position and number of disulphide bridges is similarly arbitrary, the only requirement being that CIC,ab and /I are covalently linked. The scheme for the breakdown of a” and the designation of the fragments as C3e and C3d is tentative.
The /? chain is resistant to proteolysis throughout this incubation. The splits seen are shown schematically in Fig. 3. (2) The requirement for C3b ZNA The possibility that the proteolysis of the adcfb chain seen above was nonC3b INA-dependent, being caused by either /?lH or possibly by traces of proteolytic enzymes contained in the BlH preparation, was investigated in the following manner. Parallel incubations (at the concentrations indicated above) of C3b + j31H and C3b + C3b INA + PIH were set up and aliquots taken for SDS-PAGE at 1 and 14 hr. Figure 4 shows that 81 H by itself has no proteolytic action on C3b (tracks 1 and 2) whereas in the presence of C3b INA the expected splits to adc and ab occur (tracks 3 and 4). The 14 hr time point (track 4) also shows a decrease in the amount of Cedepresent and the appearance of new bands at 58K, 39K and 29K. (3) The effect of DFP on C3 breakdown The breakdown of C3b has previously been recognized as occurring in two stages-an initial DFP-resistant step followed by a DFP-sensitive step (Lachmann, Elias & Moffett, 1972). In order
that these might be identified, C3b, C3b INA and j?lH werealltreatedwith IO-* MDFP. Subsequent incubation of these (again at the concentrations given above) with aliquots taken at 1 and 14 hr for SDS-PAGE, showed that while the initial split of the adcfb chain to OFdcand afb, and also the subsequent trimming of the err\,chain to ab were both DFP-resistant (i.e. pre-treatment of C3b, C3b INA and j31H with DFP had no effect on these events), further breakdown of the aedcchain was not (Fig. 4, tracks 5 and 6). Further investigation of the DFP-sensitive step showed that DFP treatment of j?lH preparations alone was sufficient to inhibit CTedc breakdown. (4) The use of serum as a source of C3b ZNA and BlH
C3b (again 0.5 mg-ml-i) was incubated with fresh human serum (1 ,~l serum/l00 pg C3b) and aliquots taken for SDS-PAGE at 10 min, 100 min, 250 min and 24 hr. In a parallel incubation, j?lH (BIH:C3b 1:40 by wt) was also added. Again the c@fb band is lost, and concomitant with this loss the new bands of c+ and ab appear (Fig. 5). (The aedc chain is not seen as it runs with the albumin band). It is also apparent that the
14
R. A. HARRISON and P. J. LACHMANN
5 --
6
-d
-e
Fig. 4. The requirement for C3b INA for, and the effect of tiFP on, th; protcolysis of the C3 a chain. The reduced proteins were run on an 8.594 poiyacrylamide gel. Tracks 1and 2: C3b (0.5 mg ml- *) + /?lH (5 Pg ml-‘) after 1 and 14 hr incubation at 37°C. Tracks 3 and 4: as tracks I and 2 but with C3b INA (5 pg-ml-‘) added. Tracks 5 and 6: as tracks 3 and 4 but using DFP-treated BIH. Tracks 7 and 9: marker proteins. (a) pyruvate decarboxylase, (b) catalase, (c) o-amino acid oxidase, (d) rititol dehydrogenase, (e) myoglobin. Track 8: C3c isolated from aged serum. A low level of contamination with uncleaved C3 and also a polypeptide with a molecular weight of 85K(x) is seen. The position of C3-derived chains are indicated. The relative staining intensity of ZEand ab should be compared with that of ti + & and ab in Fig. I.
relative amounts of afb and ab bands alter during the incubation, from being roughly equal at 100 min to being mainly ab at 24 hr. In contrast to the pattern seen with purified components, it would appear that the c&c chain is relatively stable in low concentrations of serum since while a edcitself
cannot be seen, no new bands of lower mol. wt, particularly at 29K, appear, even with the 24 hr time point. The parallel incubation with added PlH shows a marked acceleration in the rate of appearance of the a chain, but again no breakdown products of the c& chain are seen.
The Third Component of Human Complement
Track 2 34 31-a
S&S
6 --i
5 -.
7
0
9
_ =aedcfb c =edcfb --6
Fig. 5. The breakdown of C3b in fresh serum. The reduced proteins were run in SDS on a 10% polyacrylamide gel. Track 1: serum at an approximately equivalent load to that in tracks 2-9. Tracks 2-5: C3b (0.5 mg ml-r) + serum (1 ~I/100 yg C3b) after 10,100,250 min and 24 hr incubation at 37°C. Tracks 6-9: as tracks 2-5 but with BlH (12.5 pg ml-r) added. Track 10: C3b at an equivalent load to that in tracks 2-9. Track 11:/31H at an equivalent load to that in tracks 6-9. The position ofC3-derived chains is indicated.
(5) The chain composition products
of the C3b breakdown
While the above experiments showed two distinct stages in the breakdown of C3b, they did not show the polypeptide composition of the products. C3b (0.5 mg-ml- ‘) was therefore incubated with C3b INA and j?lH (C3b:C3b INA: BlH 5O:l:lO by wt) or with a dilution of
fresh serum (2 &SO ,ug C3) and multiple aliquots taken at 24 hr. One aliquot of each incubation was electrophoresed in SDS under non-reducing conditions (Fig. 6). [When non-reduced, C3 and C3b frequently give rise to oligomer bands, and these are seen in the C3b used in this experiment (track 2). These disappear under reducing conditions, the characteristic ol~cfb and /I chains being the only ones visible.] With serum as a
R. A. HARRISON and P. J. LACH IMIANN
Track 3
4
Fig. 6. Electrophoresis in SDS of non-reduced C3 and its fragmentation products on a 7f% polyacrylamide gel. Track 1: pyruvate decarboxylase. Track 2: C3b at a loading equivalent to that in tracks 3 and 4. Track 3: C3b (0.5 mg ml-*) i- C3b INA (10 ~(gml-i) + non-DFP-treated j1H (100 pg ml-i) after incubation at 37”Cfor24hr.Track4: as track 3 but with serum(2~1/50~gC3b)replacingC3bandINA/?1H. Track 5: BlH at a loading equivalent to that in tracks 3 and 4. Track 6: Serum at a loading equivalent to that in track 4. The position of C3 and its products are indicated, C3d would not be seen on a 7f% gel.
source of C3b INA and BlH, a single band, which is not resolved from the C3b band, is the only band seen. The duplicate aliquot run under reducing conditions showed only the &c. zb and p chains. Thus in the C3b INA-reacted C3b these chains are still held together by disulphide bonds.
With purified components, however, an additional band with a mobility slightly less than that of fi1H is seen. This correlated with the disappearance of Clrdcand the appearance of the 29K band when the sample was electrophoresed under reducing conditions.
17
The Third Component of Human Complement Table 1
Incubation ______ A B C D E F G H
C3b
DFP-treated U-I
5Pg 5 pg 5/Ig 5 fig -
0.2 0.2 0.2 0.2 -
/Ig pg pg l(g
DFP-treated KAF 0.2 pg 0.2 pg 0.2 peg 0.2 pg
Vol. PBS --___ 10 /ll 10 /II IO /I1 10 PI 10 PI 10 /ll 10 /I1 10 jll
Area of Factor B dependent haemolysis 0 0 0 0.90 0.92 0.96 0.96 0.92
The incubations shown were set up and left at 37°C. After 2 hr 10 pg Factor B and approximately 1 pg Factor D in a volume of 10 ~1 PBS were added. After a further 2 hr these were tested for the presence of haemolytically active Factor B.
To attempt tocorrelate these products with the classical immunochemically defined C3c (West et al., 1966), a sample of C3c purified from aged human serum was reduced and subjected to SDS-PAGE. The result, shown in Fig. 4, track 8, shows the band pattern of c+&fi. (6) Functional properties of the C3 breakdown products C3b, C3b INA-treated C3b, BlH-treated C3b and C3b INA/fil H treated C3b were all tested for the ability to consume Factor B in the presence of Factor D (Table 1). With untreated, /31Htreated, and C3b INA-treated C3b there was total depletion of Factor B, no haemolysis being seen. However, with the C3b INA/BlH-treated C3b there was little or no consumption of Factor B. DISCUS!3ION
Study of the mechanism of C3 breakdown is of importance for two reasons. Firstly it is only with a knowledge of the stable products formed during the activation and inactivation sequences of this multifunctional protein that full understanding as to the mediators of its varied functions can be obtained. Second, full structural analysis of a protein as large as C3 would be simplified if a series of large fragments derived from it could be prepared in good yield. Clearly the physiological products of C3 activation and inactivation could be used in this manner. In the work reported here, we find that the C3b INA has an absolute requirement, both for scission of the acddb chain and for abrogation of C3b functional (feedback) activity, for a cofactor. Furthermore we find that the stable product of C3b INA-dependent action on C3b (C3bi) contains two a chain fragments (68K and 43K) and the intact B chain, held together by
disulphide bridges, and that the generation of C3c and C3d from this requires an additional protease. This is in agreement with the work of others (Pangburn et al., 1977; Nagasawa & Stroud, 1977). However, while both these groups believe C3bi to arise as a result of a single scission of the a chain of C3b, we have shown two cleavage points. It is clear that both are physiological since they are seen not only with purified components, but also with serum as a source of these components. Thus, in addition to the stable a chain fragments described above, a third fragment (46K-afb) is transiently seen. The fact that there is no 71K fragment (acdcf) produced, together with the time course of appearance and disappearance of afb and of appearance ab, strongly suggests that ab is derived from afb and not by an independent scission of the Dledcfb chain. The fate of the 3K fragment (af) is unknown. Neither scission is DFP-inhibitable, unlike the breakdown of Cledc seen on prolonged incubation of C3b with C3b INA and /31H. In addition, both the formation of adc and afb from aedcfband the trimming of afb to ab are affected in a parallel fashion by /31H, in the presence of relatively large amounts of BlH the afb chain being only transiently seen. Thus, either C3b INA has two sites of action on the c&fi chain, or a non-DFP-sensitive serum protease, contained in /II H preparations (and therefore at less than 0.02% by weight with respect to C3b), acts rapidly on the afb fragment producing ab. This second hypothesis also requires that any such contaminant has no further action on the a&c-a$? product. Further data, such as sequence information around the cleavage sites, are required before it can be determined whether both scissions are due to C3b INA action or whether a second highly specific protease is involved.
R. A. HARRISON and P. J. LACHMANN a- 126K 2 c30 i (63.): 9K IOK
(C3dl 29K
ii ““li]”
3K
t]”
43K
B-75K C3bi
t t
:*
t c3c
t
Fig. 7. A schematic representation of C3 indicating the cleavage points on the a chain and the derivation of C3 breakdown products. (I) The site of C3 convertase action. (2) Splits caused by DFP-inhibitable proteases. (3) The sites of C3b INAdependent proteolysis. The intact /I chain is contained in C3, C3b, C3bi and C3c. Interchain disulphide bridges must exist between the a~, ab and /I chains, but their number and position have been arbitrarily indicated here. The identification of @ and ad as C3e and C3d respectively is tentative.
Controversy exists as to the identity of the C3b INA cofactor. While Pangbum and co-workers, using C3b prepared from C3 that had been passed through a Sepharose-anti+1 H column, found that the cofactor requirement could be satisfied with pl H, Nagasawa and Stroud ascribe the role of cofactor to a high molecular weight protein previously called the C4b inactivator cofactor (Shiraishi & Stroud, 1975; Nagasawa, Shiraishi & Stroud, 1976). Both of these groups used considerably higher amounts of the cofactor than the catalytic amounts of /?lH we report here. Since the requirement for a co-factor has only recently been recognized, it is probable that, unless it is rigorously excluded, C3 preparations generally contain low levels of the cofactor. In which case, whether it were fll H, the C3b inactivator cofactor, or possibly an as yet unidentified protein, it would have been removed by the ‘anti-impurity’ column used in our work. (C3b INA alone will not cleave highly purified C3b and the cofactor cannot therefore contaminate C3b INA preparations.) The BlH used in our work to provide the cofactor was about 98% pure, and the presence of either the C4b inactivator cofactor (or a further unidentified factor) cannot be ruled out. However, if there was a 2% contamination of the C4b inactivator with 81 H in the experiments that Nagasawa and Stroud report, this would produce a weight ratio of BlH:C3b of 1:120, giving total CLtdcfb cleavage in 30 min. These conditions are similar to those that we have reported for total SLedcfbconversion. The reciprocal argument applied to our BlH preparations would give a C4b inactivator cofactor:C3b ratio of 1:5000 (as opposed to the ratio of 1:2.5 used by Nagasawa and Stroud). The present evidence is therefore strongly
indicative of the cofactor being fi1H. How this factor, assumed to be j?lH, functions is as yet unclear. Whatever their mode of action, it is clear that both C3b INA and fl1H are required in catalytic amounts only with regard to C3b. Firstly, the data available does not permit one to say which it is that has the proteolytic activity on C3b. Whichever is the cofactor to the protease could conceivably act in one of three ways. /I 1H is already known to interact with C3b, dissociating Bb from it, and thus allowing C3b INA action to occur (Whaley & Ruddy, 1976; Weiler, Daha, Austen & Fearon, 1976). This interaction of fl1H with C3b might be required either to form C3b INA binding site or to expose the bond that is split in C3b INA-dependent manner. In this case a complex of all three proteins would be required for catalysis to occur. Such a requirement would increase the specificity of the protease. Other possible modes of action include a sequential action of the two components, or the neutralization by /?lH of a C3b INA-inhibitory factor (see Harrison 8c Lachmann, 1978) still present in the incubations. Such a factor must clearly, if it exists, be present in very low amounts since it is seen neither on SDS-polyacrylamide gels nor by immunochemical methods. However, if continual trace activation of C3 to C3b by serum enzymes is important in triggering the alternative pathway (“The tick-over hypothesis”-Nicol & between Lachmann, 1973), the balance potentiation and inhibition of C3b INA action could determine whether amplification or damping of C3 activation by fluid phase C3 convertases occurs (as with protective surfaces and bound C3b-Fearon & Auston, 1977a,b). Previous work on C3b fragmentation has shown the following sequence of events:
19
The Third Component of Human Complement
C3b + (C3bi) + C3c + C3d. The existence of C3bi as an intermediate between C3b and C3c and C3d formation was recognized in the conglutinin binding ability of C3b INA-reacted cell-bound C3. C3c generation or the release of bound C3 from cells required an additional DFP sensitive protease (Lachmann et al., 1972). We now find two intermediates before the DFPsensitive step. The @c-c&-B product occurs only transiently, and we have not been able to test it for Factor B consumption or conglutininbinding activity. However, since both in serum and with purified components, the stable product formed is aedc-ab-/I, and it is this inactive (in B consumption) component which requires DFPsensitive proteases for further breakdown, it seems correct to term it C3bi. Inability to demonstrate interaction between it and conglutinin is discussed in a following paper (Harrison & Lachmann, in press). C3c and C3d, isolated from aged serum, have been characterized as products of about 150K and 30K, respectively (Bokisch et al., 1969). We have shown C3c (i.e. the high mol. wt product isolated from aged serum) to be @-c&-j?.The further splits of @c, seen with non-DFP-treated PlH, were therefore of interest since they seemed to mimic the events occurring in serum and leading to C3c production (since the protease responsible can only be present at 0.02% by weight, a high degree of specificity is also implied). We have previously suggested (Harrison & Lachmann, 1978) that C3d. might be derived from C3c. This was because only one stable band at about 30K was seen with SDS-PAGE of fragmented C3bi. However, in addition to the stable 29K band seen on aedc breakdown, bands at 58K and 39K are also transiently seen. If these are the putative adc and ycd chains (Fig. 7. see also Fig. 3). the 29K chain seen in C3c being af, it is apparent that further breakdown would give rise to a second 29K fragment, ad. This, as well as ac, is in good agreement with the size of C3d isolated from aged serum. Failure to identify this as the probable derivation of C3d has arisen from the identical sizes and hence mobility on SDS-PAGE of ECand [email protected] hypothesis is strengthened by the release of a 10K fragment (ae) from cell bound C3b, and the subsequent formation of a 58K polypeptide (adc) in a C3b INA-like scission (Harrison R. A. & Lachmann P. J., in press). The a and j3 chains of C3 are held together by disulphide bridges (Nilsson & Mapes, 1973). Here we show that the aedc, afb or ab and j? chains are also held in disulphide linkage. The aedc
linkage is mediated through its ac portion since it is only that fragment which is seen in C3c. The putative ar fragment could either remain covalently bonded to C3bi, or be released during trimming of afi to ab. While the sum of the weights of the a chain fragments adds up to 123K, which is close to that which we find for the intact a chain (126K; Harrison & Lachmann, in press), it is also possible that further small fragments are lost during the activation and inactivation processes. Since West and co-workers first defined antigenically the activation and breakdown products of C3 found in serum, much effort in many laboratories has been spent in trying to define their molecular parameters. Until recently, this has concentrated on antigenic characterization. We and others have now started to look more precisely at the composition of these, and the manner in which they are produced. We have particularly concentrated on events that apparently mimic those occurring in vivo. Clearly more work is required to characterize unambiguously these products, the enzymes involved in their formation, and to characterize any physiological function they may have. Work to this end is currently in progress. Acknowledgements-The authors would like to thank Prof. B. S. Hartley for his encouragement during the course of this work. RAH thanks the Wellcome Trust for a fellowship.
RJZFERENCJB
Abramson N., Alper C. A., LachmannP. J., Rosen F. S. & Jandl J. H. (1971) J. lmmun. 107, 19-27. Alwr C. A.. Abramson N.. Johnston R. B.. Jr.. Jandl J. H. & Rosen F..S. (1970) J. c&. Invest. 49, 197<1985. Alper C. A., Colten H. R., Rosen F. S., Rabson A. R., Macnab G. & Gear J. S. A. (19720) Lancer ii, 1179-l 181. Alper C. A., Rosen F. S. & Lachmann P. J. (19726) Proc. hr.
Acad. Sci. U.S.A. 69,2910-2913.
Ballow M. & Cochrane C. G. (1969) J. Immun. 103.944-953. Bokisch V. A., Mtiller-Eberdard H. J. & Cochrane C. G. (1969). .I. exp. Med. 129, 1109-I 130. Cuatrecasas P. & Anfmsen C. B. (1971) In Methods in Enzynmlogy (Edited by Jakoby W. B) Voi. 22, pp. 345-378. Academic Press, New York. Da Silva W. D., Eisele J. W. & Lepow 1. H. (1967) J. exp. Med. 116, 1027-1048. Fearon D. T. & Austen K. F. (19774) J. exp. Med. M&22-33. Fearon D. T. 8z Austen K. F. (19776) Proc. natn. Acad. Sci. U.S.A. 74, 1683-1687.
Gi8li I. & Nelson R. A. Jr. (1968) Exp. Cell Res. SL45-67. Gitlin J. D., Rosen F. S. & Lachmann P. J. (1975) J. exp. Med. 141, 1221-1226. Goodkofsky I., Stewart A. M. & Lepow 1. H. (1973) J. Immun. 111, 287. Harrison R. A. (1974) Ph.D. dissertation, University of Cambridge. Harrison R. A. & Lachmann P. J. (1978) J. Immun. 120,1777 (Abstract).
20
R. A. HARRISON and P. J. LACHMANN
Harrison R. A. & Lachmann P. J. (1979) Molec. Immun. 16, 767-776. Harrison R. A. & Lachmann P. J. Molec. Immunol.. in press. Henson P. M. (1969) Immunology._. 16. 107-121. Huber H., Poiley M. J.. Linscott W. D.. Fudenberg H. H. & Muller-Eberhard H. J. (1968) Science 162, 1281-1283. Johnston R. B. Jr., Klemperer M. R., Alper C. A. & Rosen F. S. (1969)J. e.rp. Med. 129, 1275-1290. Lachmann P. J., Elias D. E. & Moffett A. (1972) In Biological Activities of Complement (Edited by Ingram D. G.) pp. 202-214. Karger. Basel. Lachmann P. J., Hobart M. J. & Aston W. P. (1973) In Handbook oJ’ Experimental Immunology (Edited by Weir D. M.) 2nd Edn. Blackwell Scientific Publications, Oxford. Lachmann P. J. & Miiller-Eberhard H. J. (1967) In Protides oj’the Biological Fluids, Vol. 15, pp. 469-470. Elsevier, New York. Lachmann P. J. & Miiller-Eberhard H. J. (1968) J. Immun. 100, 691-698. Lachmann P. J., Nicol P. A. E. & Aston W. P. (1973) Immunochemistry 10, 695-700. Laemmli U. K. (1970) Nature, Lond. 227, 680-685. Martin A., Lachmann P. J., Halbwachs L. & Hobart M. J. (1976) Immunochemis[ry 13, 317-324. Miiller-Eberhard H. J., Dalmasso A. P. & Calcott M. A. (l966a) J. exp. Med. 123, 33-54. Mtiller-Eberhard H. J., Dalmasso A. P. & Calcott M. A. (19696) J. exp. Med. 125, 359-380.
Miiller-Eberhard H. J. & G&e 0. (1972) J. e.\-p. Med. 135, 1003-1008. Nagasawa S., Shiraishi S. & Stroud R. M. (1976) J. Immun. 116, 1743.
Nagasawa S. & Stroud R. M. (1977)
Immunochemistry
14,
Nelson R. A. Jr. (1953) Science 118, 733-737. Nicol P. A. E. & Lachmann P. J. (1973) Immunology
24,
749-756.
259-275.
Nilsson U. & Mapes J. (1973) J. fmmun. 111, 293-294. Pangburn M. K., Schreiber R. D. & Miiller-Eberhard H. J. (1977) J. e-up. Med. 146, 257-270. Ruddy S. & Austen K. F. (1971) J. Immun. 107, 742-750. Shiraishi S. & Stroud R. M. (1975) Immunochemrsrry 12, 935-939.
Tamura N. & Nelson R. A. (1967) J. Immun. 99, 582-589. Thompson R. A. & Lachmann P. J. (1977) Cfin. e.up. Immunol.
27, 23-29.
Vogt W., Schmidt G., von Buttlar B. & Dieminger L. (1978)
Immunology 34,29-40. Whaley K. & Ruddy S. (1976). Science 193, 101I-1013. Weiler J. M., Daha M. R., Austen K. F. & Fearon D. T. (1976) Proc. nom. Acad. Sci. U.S.A. 73, 3268-3272. West C. D., Davis N. C., Forristal F., Herbst J. & Spitzel R. (1966) J. Immun. 96, 650-658.
Immunology* Vol 17, pp. 9-20. Press Ltd. 1980. Printed m Great tWitam.
THE PHYSIOLOGICAL BREAKDOWN OF THE THIRD COMPONENT OF HUMAN COMPLEMENT R. A. HARRISON
and P. J. LACHMANN
Department of Biochemistry, Imperial College, London SW7, and MRC Group on Mechanisms in Tumour Immunity, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England (First received 5 January
1979; in revisedfbrm
2 April 1979)
Abstract-The C3b INA*-dependent breakdown of fluid phase C3b has been shown to have an absolute requirement for a second factor. This factor is contained in catalytic amounts (with respect to C3b) of highly purified pl H. bl H alone does not cause proteolysis of C3b. In the presence of C3b INA and BlH, proteolysis of the larger? polypeptide chain (116K) of C3b occurs. Initially, a single scission gives two chains of 68K and 46K. A subsequent split of the 46K chain yields a smaller product of 43K. All of these chains remain covalently bonded to the /I chain of C3b. These initial reactions are the same whether purified components or whether low conmntrations of serum as a source of C3b INA and /31H are used. Pre-treatment of C3b, C3b INA and BIH with DFP has no effect on these events. Further protelysis of the 68K chain requires a DFP-sensitive protease and leads-to the formation of an additional high molecular weight breakdown product of C3. This has the same polypeptide chain composition as the high molecular weight breakdown product of C3 purified from aged serum, and is therefore identified as C3c.
NOMENCLATURE
Firstly, in its nascent state, it is able to bind to a wide variety of surfaces. Binding of C3b in this manner, which does not require a specific C3b receptor, confers the so-called C3 adherence reactions to the C3b binding particle. Immune (Nelson, 1953; Henson, 1969) and opsonic (Gigli & Nelson, 1968; Johnston et al., 1969) adherence are important in facilitating ingestion of this particle by phagoyctic cells that have C3b receptors on their surface (Huber et al., 1968). Secondly, the association of C3b with either the classical (Miiller-Eberhard, Dalmasso & Calcott, 1966a, b) or the alternative pathway (Vogt et al., 1978) C3 convertase gives rise to C5 convertase activity, with consequent deposition of C5b and assembly of the late acting components C6-C9 on adjacent surfaces. Thirdly, C3b itself is an intrinsic component of the alternative pathway C3 convertase (MiillerEberhard & G&e, 1972; Goodkofsky, Stewart & Lepow, 1973). This ability of C3b to amplify its own production [the so-called C3b feedback loop (Nicol & Lachmann, 1973)] clearly requires a specific homeostatic mechanism, which is provided by the C3b INA-dependent inactivation of C3b (Miiller-Eberhard & Giitze, 1972; Alper, Rosen & Lachmann, 1972). Thus, the rate at which C3b is generated by C3 convertase activity must exceed its C3b INAdependent inactivation before amplification of C3b formation is seen. Without such a system, any adventitious activation of C3 would be
Where applicable, the nomenclature agreed by the WHO Committee on Complement has been used (Bull. W.H.O. 39, 1968). INTRODUCTION
occupies a central position within the complement cascade. Following activation by a C3 convertase, it is split into two separate molecules. C3a, the smaller, is a polypeptide anaphylatoxin (da Silva, Eisele & Lepow, 1967; Bokisch, Miiller-Eberhard & Cochrane, 1969). C3b, the larger, mediates at least three major biological functions.
C3
*Abbreviationsused: C3b INA, C3b/C4b inactivator; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; CVF, cobra venom factor; PBS, phosphate buffered saline, pH 7.2; DFP, diisopropyl fluorophosphate; 2-ME, 2-mercaptoethanol; CNBr. cyanogen bromide; K. 1000 daltons. t Fragmentation of chains: C3 is composed of two polypeptide chains, previously designated a and j?, held together by disulphide bonds and non-covalent forces (Nilsson & Mapes, 1973). In the absence of an agreed nomenclature dealing with the highly complex fragmentation of the C3 a chains we have adopted a system that facilitates identification of the a-chain fragment with the trivial name for the C3 product of which it is part. Thus, the a chain of C3 contains six physiologically distinguishable portions and is written t(ddi: c3a is aa, C3d is ad, and the at portion is tentatively identified as C3e (Harrison R. A. & Lachmann P. J., in press). The presence of disulphide bridges between separate polypeptide chains is indicated by a hyphen. Native C3 is therefore c@c~B. Designation of a product as a+@+(C3c) implies only that the three chains are disulphide-bonded in an unspecified manner. 9
IO
R. A. HARRISON
and P. J. LACHMANN
expected to lead to rapid depletion of C3 and Factor B levels in serum. Genetic deficiency of C3b INA (Alper et al., 1970; Abramson et al., 1971; Thompson & Lachmaim, 1977) as well as experimental depletion of C3b INA from serum (Nicol & Lachmann, 1973) has shown this to be the case. The severe clinical consequences of C3b INA deficiency and of C3 deficiency (Alper et al., 1972) also serve to emphasize the importance of C3-mediated functions and their regulation. While the existence of C3b INA has been known for some time (Tamura & Nelson, 1967; Lachmann & Miiller-Eberhard, 1967,1968), until recently little has been known about its action on C3b at a molecular level. Early work showed that C3b INA was enzymic in nature, not being depleted during its reaction on C3b, and also that subsequent to C3b INA action on C3b, resistance to ‘trypsin-like’ enzymes was lost (Lachmann & Miiller-Eberhard, 1968). Other workers (Ruddy & Austen, 1971) suggested that C3b INA action on C3b leads directly to the production of C3c and C3d, two antigenically defined products of C3 breakdown found in aged serum. This, however, was in conflict with the known functional stages in C3b breakdown, i.e. the acquisition of conglutinin binding activity subsequent to C3b INA action but prior to the appearance of C3c (Lachmann, Elias & Moffett, 1972). Recent work has confirmed that C3c is not formed directly by C3b INA action on C3b, at least one intermediate molecular form being demonstrable (Pangburn, Schreiber & MiillerEberhard, 1977; Nagasawa & Stroud, 1977; Harrison & Lachmann, 1978). This work also showed that C3b INA by itself had no action on C3b, a co-factor being required for proteolysis to occur. We have been studying the activation and breakdown of human C3 using both purified serum components, and also serum as a source of these components, in order to elucidate the nature of possible intermediate products. In this paper the conditions under which some of these are produced, and an initial characterization of their molecular nature is given. A preliminary report of this work has been presented elsewhere (Harrison & Lachmann, 1978). MATERIALS AND METHODS
Complement
proteins
and /ll H were purified as described (Harrison & Lachmann, 1979). In some experiments, C3 that had not been passed C3
through an anti-impurity column was used. and where applicable this is indicated. Two different preparations of /?lH were used. One was about 98% pure as adjudged by SDS-PAGE, and elicited a monospecific antibody response in rabbits. The second was about 950; pure as adjudged by staining intensity on SDS-PAGE. C3c was purified from aged human serum using the method described for C3. C3b INA was purified as described by Lachmann, Nicol and Aston (1973). Factors B and D were purified essentially as described by Lachmann, Hobart and Aston (1973). Serum was freshly drawn by venepuncture from healthy donors when required, the blood being allowed to clot for 4 hr at 22°C. Preparation
of insolubilized
CVF
CVF was prepared from Naja naja kaonthia venom (Sigma) essentially’ as described by Ballow & Cochrane (1969). Purified CVF was then dialysed against 0.2 A4 citrate, pH 6.5, its concentration adjusted to 2 mg-ml- l and the CVF coupled to Sepharose CL-4B by the method of Cuatrecasas & Anfinsen (1971) under conditions described previously (Harrison & Lachmann, 1979). Preparation
of C3b
C3b was prepared from C3 by means of an insolubilized CVF-convertase essentially as described by Gitlin et al. (1975). In early experiments C3b was separated from C3a by chromatography at pH 7.2in l.OMKClandat4”Con hydroxylapatite. C3a was unadsorbed in 0.025 M phosphate, and C3b was eluted with a linear gradient from 0.025 M to 0.2 Mphosphate. C3b, at 1 mg-ml- l, was stored frozen at -20°C in 25 mM phosphate, pH 7.0, until required. Subsequent experiments showed that dissociation and removal of C3a from C3b had no effect on the breakdown of C3b and this step was discarded. DFP treatment
of protein
DFP (Sigma) was kept at -20°C as a stock solution of 1.0 M in propan-2-01 [previously dried over a molecular sieve, type 3A (BDH)]. Solutions were made 10 - * A4with DFP and held at 22°C for 1 hr and then at 2°C for 16 hr before being stored frozen at -20°C until required. Factor B activity
Factor B was measured haemolytically by the radial diffusion method of Martin et al. (1976).
The Third Component of Human Complement
11
~hrorna~o~~aphi~ media
General in~u~a~i~n and sampling procedure
DEAE-cellulose (Whatman), Sephadex and Sepharose CL-4B (Pharmacia) were used. Hydroxylapatite was manufactured as described previously (Harrison & Lachmann, 1979).
All incubations were carried out at 37°C in PBS and in capped 2 ml glass vials (Flow taboratories). At the times indicated in the text aliquots were taken into 2 ml Pyrex tubes and
gedcfb _
i
Fig. I _ The &a% of added pl H on the C3b INAdependent c&wage of C3b. The reduced proteins were rnn in SDS on an 8.5% polyacrylamide gel. Track 1: C3b INA at a loading equivalent to that in tracks 3-10. Track 2: C3b at a loading equivalent to that in tracks 3-10. Tracks 3-6: C3b (0.5 mg ml- r) + C3b INA (5 pg ml-l) after 2,20,180 mins and 24 hr incubation at 37°C. Tracks 7-10: as tracks 3-6 but with the addition of 81 H at 5 r;lgmt - *. Tracks I I: marker proteins (approximately 5 rg of each) (a) pyruvate decarboxylase, (b) catalase, (c) n-amino acid ox&se. The position of C3derived polypeptide chains is indited.
12
R. A. HARRISON
and P. J. LACHMANN
freeze-dried immediately. The freeze-dried samples were then kept at - 20°C until analysed by SDS-PAGE. If samples were required for immunochemical characterization they were frozen at -20°C immediately upon aliquoting and stored at -20°C until required.
Harrison, 1974), and ribitol dehydrogenase (Klebsiella aerogenes, 27K) (the gift of Dr. J. M. Dothie). RESULTS
(1) The requirement for a cofactor (p 1H)
SDS polyacrylamide gel electrophoresis
SDS-PAGE was carried out on polyacrylamide slabs using the buffers described by Laemmli (1970). Samples were dissolved in either 25 ~1 8.0 M urea, 1’;: mercaptoethanol, l”/, SDS (if the reduced sample was to be observed), or 25 ,uI 8.0 M urea, 19; SDS (for analysis of the nonreduced protein), and heated at 100°C for 2 min prior to loading. The final acrylamide concentration in the resolving gel varied and is indicated in the text. Gels were stained with 0.2% Coomassie Brilliant Blue G250 (Lamb). Marker proteins used were RNA polymerase from E. coli (B chain 165K, j?’ chain 155K, c1 39K), catalase (beef liver, 58K), D-amino acid oxidase (Hog kidney, 37K), myoglobin (sperm whale, 17.8K), and lysozyme (egg white, 14.3K) (all from Sigma), pyruvate decarboxylase, the E, component of the pyruvate dehydrogenase multi-enzyme complex of E. coli (103K-
lo0.000
-
40.000
-
Two parallel incubations were set up. In the first, C3b (0.5 mg-ml- ‘) was mixed with C3b INA (C3b INA:C3b 1:100 by wt). In the second, j?lH (j?lH:C3b 1:lOO by wt) was also included. Both were left for 24 hr, aliquots being taken for SDS-PAGE at 2 min, 20 min, 3 hr and 24 hr. As can be seen in Fig. 1, no cleavage of either the u (aedcfb)or fl chain of C3b occurred in the absence of PlH. In the presence of catalytic amounts of PlH, however, the 20 min and 3 hr time points showed three new bands at 68K (c&c), 46K (~fb) and 43K (&). As these appear the cledcfbchain is lost. The time course of their appearance together with their size suggests strongly that the CLedcfb chain is initially split into Cpdcand ~(fi, and that subsequent ‘trimming’ of the clfb chain produces c&, a small 3K peptide (af) being lost. During prolonged incubation (24 hr) in the presence of fil H (Fig. 1, track IO) the @dcchain is further fragmented and a succession of smaller polypeptides culminating in one at 29K are seen.
i P t B a 2 i”
61.
\
7-m \
lO.OOOl
I 02
I 04
I
I
06
0.6
Mobility Fig. 2. The molecular nature ofC3 arham-derived polypeptidechams. C3 fragmentation products were run gel and mobilities relative to bromphenol blue under reducing conditions on a 12”” SDS-polyacrylamide measured. Marker proteins used were: (1) RNA polymerase B chain; (2) RNA polymerase 8’ chain; (3) pyruvate decarboxylase; (4) catalase; (5) o-amino acid oxidase; (6) ribitol dehydrognease; (7) myoglobulin. The mobilities of C3-derived polypeptide chains are indicated by arrows. (a) a*edcth:126K; (b) aed? 1 l7K; (c) p: 75K: (d) a+ 68K: (e) a% 58K; (0 all: 46K; (g) ab: 43K; (h) a=? 39K: (i) a’ + (ad): 29K.
13
The Third Component of Human Complement
I’
aO+
C3a t C3b
C3a
I
i
a:+
t ab
C3bi
I
(C3d) (C3e) Fig. 3. Schematic representation of the breakdown of C3. (1) C3 convertase-dependent cleavage; (2) C3b INAdependent cleavage; (3) DFP-inhibitable cleavage. Arrows indicate the cleavage points. The position indicated for the a( fragment is arbitrary and it could equally well lie at the opposite end of the ab chain. It is also possible that it remains disulphide-linked to one of the chains in C3c. The interchain position and number of disulphide bridges is similarly arbitrary, the only requirement being that CIC,ab and /I are covalently linked. The scheme for the breakdown of a” and the designation of the fragments as C3e and C3d is tentative.
The /? chain is resistant to proteolysis throughout this incubation. The splits seen are shown schematically in Fig. 3. (2) The requirement for C3b ZNA The possibility that the proteolysis of the adcfb chain seen above was nonC3b INA-dependent, being caused by either /?lH or possibly by traces of proteolytic enzymes contained in the BlH preparation, was investigated in the following manner. Parallel incubations (at the concentrations indicated above) of C3b + j31H and C3b + C3b INA + PIH were set up and aliquots taken for SDS-PAGE at 1 and 14 hr. Figure 4 shows that 81 H by itself has no proteolytic action on C3b (tracks 1 and 2) whereas in the presence of C3b INA the expected splits to adc and ab occur (tracks 3 and 4). The 14 hr time point (track 4) also shows a decrease in the amount of Cedepresent and the appearance of new bands at 58K, 39K and 29K. (3) The effect of DFP on C3 breakdown The breakdown of C3b has previously been recognized as occurring in two stages-an initial DFP-resistant step followed by a DFP-sensitive step (Lachmann, Elias & Moffett, 1972). In order
that these might be identified, C3b, C3b INA and j?lH werealltreatedwith IO-* MDFP. Subsequent incubation of these (again at the concentrations given above) with aliquots taken at 1 and 14 hr for SDS-PAGE, showed that while the initial split of the adcfb chain to OFdcand afb, and also the subsequent trimming of the err\,chain to ab were both DFP-resistant (i.e. pre-treatment of C3b, C3b INA and j31H with DFP had no effect on these events), further breakdown of the aedcchain was not (Fig. 4, tracks 5 and 6). Further investigation of the DFP-sensitive step showed that DFP treatment of j?lH preparations alone was sufficient to inhibit CTedc breakdown. (4) The use of serum as a source of C3b ZNA and BlH
C3b (again 0.5 mg-ml-i) was incubated with fresh human serum (1 ,~l serum/l00 pg C3b) and aliquots taken for SDS-PAGE at 10 min, 100 min, 250 min and 24 hr. In a parallel incubation, j?lH (BIH:C3b 1:40 by wt) was also added. Again the c@fb band is lost, and concomitant with this loss the new bands of c+ and ab appear (Fig. 5). (The aedc chain is not seen as it runs with the albumin band). It is also apparent that the
14
R. A. HARRISON and P. J. LACHMANN
5 --
6
-d
-e
Fig. 4. The requirement for C3b INA for, and the effect of tiFP on, th; protcolysis of the C3 a chain. The reduced proteins were run on an 8.594 poiyacrylamide gel. Tracks 1and 2: C3b (0.5 mg ml- *) + /?lH (5 Pg ml-‘) after 1 and 14 hr incubation at 37°C. Tracks 3 and 4: as tracks I and 2 but with C3b INA (5 pg-ml-‘) added. Tracks 5 and 6: as tracks 3 and 4 but using DFP-treated BIH. Tracks 7 and 9: marker proteins. (a) pyruvate decarboxylase, (b) catalase, (c) o-amino acid oxidase, (d) rititol dehydrogenase, (e) myoglobin. Track 8: C3c isolated from aged serum. A low level of contamination with uncleaved C3 and also a polypeptide with a molecular weight of 85K(x) is seen. The position of C3-derived chains are indicated. The relative staining intensity of ZEand ab should be compared with that of ti + & and ab in Fig. I.
relative amounts of afb and ab bands alter during the incubation, from being roughly equal at 100 min to being mainly ab at 24 hr. In contrast to the pattern seen with purified components, it would appear that the c&c chain is relatively stable in low concentrations of serum since while a edcitself
cannot be seen, no new bands of lower mol. wt, particularly at 29K, appear, even with the 24 hr time point. The parallel incubation with added PlH shows a marked acceleration in the rate of appearance of the a chain, but again no breakdown products of the c& chain are seen.
The Third Component of Human Complement
Track 2 34 31-a
S&S
6 --i
5 -.
7
0
9
_ =aedcfb c =edcfb --6
Fig. 5. The breakdown of C3b in fresh serum. The reduced proteins were run in SDS on a 10% polyacrylamide gel. Track 1: serum at an approximately equivalent load to that in tracks 2-9. Tracks 2-5: C3b (0.5 mg ml-r) + serum (1 ~I/100 yg C3b) after 10,100,250 min and 24 hr incubation at 37°C. Tracks 6-9: as tracks 2-5 but with BlH (12.5 pg ml-r) added. Track 10: C3b at an equivalent load to that in tracks 2-9. Track 11:/31H at an equivalent load to that in tracks 6-9. The position ofC3-derived chains is indicated.
(5) The chain composition products
of the C3b breakdown
While the above experiments showed two distinct stages in the breakdown of C3b, they did not show the polypeptide composition of the products. C3b (0.5 mg-ml- ‘) was therefore incubated with C3b INA and j?lH (C3b:C3b INA: BlH 5O:l:lO by wt) or with a dilution of
fresh serum (2 &SO ,ug C3) and multiple aliquots taken at 24 hr. One aliquot of each incubation was electrophoresed in SDS under non-reducing conditions (Fig. 6). [When non-reduced, C3 and C3b frequently give rise to oligomer bands, and these are seen in the C3b used in this experiment (track 2). These disappear under reducing conditions, the characteristic ol~cfb and /I chains being the only ones visible.] With serum as a
R. A. HARRISON and P. J. LACH IMIANN
Track 3
4
Fig. 6. Electrophoresis in SDS of non-reduced C3 and its fragmentation products on a 7f% polyacrylamide gel. Track 1: pyruvate decarboxylase. Track 2: C3b at a loading equivalent to that in tracks 3 and 4. Track 3: C3b (0.5 mg ml-*) i- C3b INA (10 ~(gml-i) + non-DFP-treated j1H (100 pg ml-i) after incubation at 37”Cfor24hr.Track4: as track 3 but with serum(2~1/50~gC3b)replacingC3bandINA/?1H. Track 5: BlH at a loading equivalent to that in tracks 3 and 4. Track 6: Serum at a loading equivalent to that in track 4. The position of C3 and its products are indicated, C3d would not be seen on a 7f% gel.
source of C3b INA and BlH, a single band, which is not resolved from the C3b band, is the only band seen. The duplicate aliquot run under reducing conditions showed only the &c. zb and p chains. Thus in the C3b INA-reacted C3b these chains are still held together by disulphide bonds.
With purified components, however, an additional band with a mobility slightly less than that of fi1H is seen. This correlated with the disappearance of Clrdcand the appearance of the 29K band when the sample was electrophoresed under reducing conditions.
17
The Third Component of Human Complement Table 1
Incubation ______ A B C D E F G H
C3b
DFP-treated U-I
5Pg 5 pg 5/Ig 5 fig -
0.2 0.2 0.2 0.2 -
/Ig pg pg l(g
DFP-treated KAF 0.2 pg 0.2 pg 0.2 peg 0.2 pg
Vol. PBS --___ 10 /ll 10 /II IO /I1 10 PI 10 PI 10 /ll 10 /I1 10 jll
Area of Factor B dependent haemolysis 0 0 0 0.90 0.92 0.96 0.96 0.92
The incubations shown were set up and left at 37°C. After 2 hr 10 pg Factor B and approximately 1 pg Factor D in a volume of 10 ~1 PBS were added. After a further 2 hr these were tested for the presence of haemolytically active Factor B.
To attempt tocorrelate these products with the classical immunochemically defined C3c (West et al., 1966), a sample of C3c purified from aged human serum was reduced and subjected to SDS-PAGE. The result, shown in Fig. 4, track 8, shows the band pattern of c+&fi. (6) Functional properties of the C3 breakdown products C3b, C3b INA-treated C3b, BlH-treated C3b and C3b INA/fil H treated C3b were all tested for the ability to consume Factor B in the presence of Factor D (Table 1). With untreated, /31Htreated, and C3b INA-treated C3b there was total depletion of Factor B, no haemolysis being seen. However, with the C3b INA/BlH-treated C3b there was little or no consumption of Factor B. DISCUS!3ION
Study of the mechanism of C3 breakdown is of importance for two reasons. Firstly it is only with a knowledge of the stable products formed during the activation and inactivation sequences of this multifunctional protein that full understanding as to the mediators of its varied functions can be obtained. Second, full structural analysis of a protein as large as C3 would be simplified if a series of large fragments derived from it could be prepared in good yield. Clearly the physiological products of C3 activation and inactivation could be used in this manner. In the work reported here, we find that the C3b INA has an absolute requirement, both for scission of the acddb chain and for abrogation of C3b functional (feedback) activity, for a cofactor. Furthermore we find that the stable product of C3b INA-dependent action on C3b (C3bi) contains two a chain fragments (68K and 43K) and the intact B chain, held together by
disulphide bridges, and that the generation of C3c and C3d from this requires an additional protease. This is in agreement with the work of others (Pangburn et al., 1977; Nagasawa & Stroud, 1977). However, while both these groups believe C3bi to arise as a result of a single scission of the a chain of C3b, we have shown two cleavage points. It is clear that both are physiological since they are seen not only with purified components, but also with serum as a source of these components. Thus, in addition to the stable a chain fragments described above, a third fragment (46K-afb) is transiently seen. The fact that there is no 71K fragment (acdcf) produced, together with the time course of appearance and disappearance of afb and of appearance ab, strongly suggests that ab is derived from afb and not by an independent scission of the Dledcfb chain. The fate of the 3K fragment (af) is unknown. Neither scission is DFP-inhibitable, unlike the breakdown of Cledc seen on prolonged incubation of C3b with C3b INA and /31H. In addition, both the formation of adc and afb from aedcfband the trimming of afb to ab are affected in a parallel fashion by /31H, in the presence of relatively large amounts of BlH the afb chain being only transiently seen. Thus, either C3b INA has two sites of action on the c&fi chain, or a non-DFP-sensitive serum protease, contained in /II H preparations (and therefore at less than 0.02% by weight with respect to C3b), acts rapidly on the afb fragment producing ab. This second hypothesis also requires that any such contaminant has no further action on the a&c-a$? product. Further data, such as sequence information around the cleavage sites, are required before it can be determined whether both scissions are due to C3b INA action or whether a second highly specific protease is involved.
R. A. HARRISON and P. J. LACHMANN a- 126K 2 c30 i (63.): 9K IOK
(C3dl 29K
ii ““li]”
3K
t]”
43K
B-75K C3bi
t t
:*
t c3c
t
Fig. 7. A schematic representation of C3 indicating the cleavage points on the a chain and the derivation of C3 breakdown products. (I) The site of C3 convertase action. (2) Splits caused by DFP-inhibitable proteases. (3) The sites of C3b INAdependent proteolysis. The intact /I chain is contained in C3, C3b, C3bi and C3c. Interchain disulphide bridges must exist between the a~, ab and /I chains, but their number and position have been arbitrarily indicated here. The identification of @ and ad as C3e and C3d respectively is tentative.
Controversy exists as to the identity of the C3b INA cofactor. While Pangbum and co-workers, using C3b prepared from C3 that had been passed through a Sepharose-anti+1 H column, found that the cofactor requirement could be satisfied with pl H, Nagasawa and Stroud ascribe the role of cofactor to a high molecular weight protein previously called the C4b inactivator cofactor (Shiraishi & Stroud, 1975; Nagasawa, Shiraishi & Stroud, 1976). Both of these groups used considerably higher amounts of the cofactor than the catalytic amounts of /?lH we report here. Since the requirement for a co-factor has only recently been recognized, it is probable that, unless it is rigorously excluded, C3 preparations generally contain low levels of the cofactor. In which case, whether it were fll H, the C3b inactivator cofactor, or possibly an as yet unidentified protein, it would have been removed by the ‘anti-impurity’ column used in our work. (C3b INA alone will not cleave highly purified C3b and the cofactor cannot therefore contaminate C3b INA preparations.) The BlH used in our work to provide the cofactor was about 98% pure, and the presence of either the C4b inactivator cofactor (or a further unidentified factor) cannot be ruled out. However, if there was a 2% contamination of the C4b inactivator with 81 H in the experiments that Nagasawa and Stroud report, this would produce a weight ratio of BlH:C3b of 1:120, giving total CLtdcfb cleavage in 30 min. These conditions are similar to those that we have reported for total SLedcfbconversion. The reciprocal argument applied to our BlH preparations would give a C4b inactivator cofactor:C3b ratio of 1:5000 (as opposed to the ratio of 1:2.5 used by Nagasawa and Stroud). The present evidence is therefore strongly
indicative of the cofactor being fi1H. How this factor, assumed to be j?lH, functions is as yet unclear. Whatever their mode of action, it is clear that both C3b INA and fl1H are required in catalytic amounts only with regard to C3b. Firstly, the data available does not permit one to say which it is that has the proteolytic activity on C3b. Whichever is the cofactor to the protease could conceivably act in one of three ways. /I 1H is already known to interact with C3b, dissociating Bb from it, and thus allowing C3b INA action to occur (Whaley & Ruddy, 1976; Weiler, Daha, Austen & Fearon, 1976). This interaction of fl1H with C3b might be required either to form C3b INA binding site or to expose the bond that is split in C3b INA-dependent manner. In this case a complex of all three proteins would be required for catalysis to occur. Such a requirement would increase the specificity of the protease. Other possible modes of action include a sequential action of the two components, or the neutralization by /?lH of a C3b INA-inhibitory factor (see Harrison 8c Lachmann, 1978) still present in the incubations. Such a factor must clearly, if it exists, be present in very low amounts since it is seen neither on SDS-polyacrylamide gels nor by immunochemical methods. However, if continual trace activation of C3 to C3b by serum enzymes is important in triggering the alternative pathway (“The tick-over hypothesis”-Nicol & between Lachmann, 1973), the balance potentiation and inhibition of C3b INA action could determine whether amplification or damping of C3 activation by fluid phase C3 convertases occurs (as with protective surfaces and bound C3b-Fearon & Auston, 1977a,b). Previous work on C3b fragmentation has shown the following sequence of events:
19
The Third Component of Human Complement
C3b + (C3bi) + C3c + C3d. The existence of C3bi as an intermediate between C3b and C3c and C3d formation was recognized in the conglutinin binding ability of C3b INA-reacted cell-bound C3. C3c generation or the release of bound C3 from cells required an additional DFP sensitive protease (Lachmann et al., 1972). We now find two intermediates before the DFPsensitive step. The @c-c&-B product occurs only transiently, and we have not been able to test it for Factor B consumption or conglutininbinding activity. However, since both in serum and with purified components, the stable product formed is aedc-ab-/I, and it is this inactive (in B consumption) component which requires DFPsensitive proteases for further breakdown, it seems correct to term it C3bi. Inability to demonstrate interaction between it and conglutinin is discussed in a following paper (Harrison & Lachmann, in press). C3c and C3d, isolated from aged serum, have been characterized as products of about 150K and 30K, respectively (Bokisch et al., 1969). We have shown C3c (i.e. the high mol. wt product isolated from aged serum) to be @-c&-j?.The further splits of @c, seen with non-DFP-treated PlH, were therefore of interest since they seemed to mimic the events occurring in serum and leading to C3c production (since the protease responsible can only be present at 0.02% by weight, a high degree of specificity is also implied). We have previously suggested (Harrison & Lachmann, 1978) that C3d. might be derived from C3c. This was because only one stable band at about 30K was seen with SDS-PAGE of fragmented C3bi. However, in addition to the stable 29K band seen on aedc breakdown, bands at 58K and 39K are also transiently seen. If these are the putative adc and ycd chains (Fig. 7. see also Fig. 3). the 29K chain seen in C3c being af, it is apparent that further breakdown would give rise to a second 29K fragment, ad. This, as well as ac, is in good agreement with the size of C3d isolated from aged serum. Failure to identify this as the probable derivation of C3d has arisen from the identical sizes and hence mobility on SDS-PAGE of ECand [email protected] hypothesis is strengthened by the release of a 10K fragment (ae) from cell bound C3b, and the subsequent formation of a 58K polypeptide (adc) in a C3b INA-like scission (Harrison R. A. & Lachmann P. J., in press). The a and j3 chains of C3 are held together by disulphide bridges (Nilsson & Mapes, 1973). Here we show that the aedc, afb or ab and j? chains are also held in disulphide linkage. The aedc
linkage is mediated through its ac portion since it is only that fragment which is seen in C3c. The putative ar fragment could either remain covalently bonded to C3bi, or be released during trimming of afi to ab. While the sum of the weights of the a chain fragments adds up to 123K, which is close to that which we find for the intact a chain (126K; Harrison & Lachmann, in press), it is also possible that further small fragments are lost during the activation and inactivation processes. Since West and co-workers first defined antigenically the activation and breakdown products of C3 found in serum, much effort in many laboratories has been spent in trying to define their molecular parameters. Until recently, this has concentrated on antigenic characterization. We and others have now started to look more precisely at the composition of these, and the manner in which they are produced. We have particularly concentrated on events that apparently mimic those occurring in vivo. Clearly more work is required to characterize unambiguously these products, the enzymes involved in their formation, and to characterize any physiological function they may have. Work to this end is currently in progress. Acknowledgements-The authors would like to thank Prof. B. S. Hartley for his encouragement during the course of this work. RAH thanks the Wellcome Trust for a fellowship.
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