C3, C4, and α2M share the unusual structural feature of an intrachain thioester bond, which enables them to bind covalently to âtargetâ molecules (2â 4).
The Covalent Binding Reaction of Complement Component C31 Mihaela Gadjeva,2* Alister W. Dodds,* Aiko Taniguchi-Sidle,† Antony C. Willis,* David E. Isenman,† and S. K. Alex Law3* The covalent binding of C3 to target molecules on the surfaces of pathogens is crucial in most complement-mediated activities. When C3 is activated, the acyl group is transferred from the sulfhydryl of the internal thioester to the hydroxyl group of the acceptor molecule; consequently, C3 is bound to the acceptor surface by an ester bond. It has been determined that the binding reaction of the B isotype of human C4 uses a two-step mechanism. Upon activation, a His residue first attacks the internal thioester to form an acyl-imidazole bond. The freed thiolate anion of the Cys residue of the thioester then acts as a base to catalyze the transfer of the acyl group from the imidazole to the hydroxyl group of the acceptor molecule. In this article, we present results which indicate that this two-step reaction mechanism also occurs in C3. The Journal of Immunology, 1998, 161: 985–990.
C
omplement components C3, C4, C5, and the proteinase inhibitor a2-macroglobulin (a2M)4 constitute a family of proteins with a high degree of sequence similarity and similar gene organization. They are thought to have arisen by gene duplication from a common ancestor (1). C3, C4, and a2M share the unusual structural feature of an intrachain thioester bond, which enables them to bind covalently to “target” molecules (2– 4). Upon proteolytic activation, the thioester becomes “exposed” on the surface of the molecule and reactive with nucleophiles. Ultimately, a covalent bond is formed between the acyl group of the thioester and the amino or hydroxyl groups of the target. The thioester proteins show significant differences in their binding preferences. C3 and the C4B isotype of human C4 predominantly form ester bonds with hydroxyl groups on carbohydrates or proteins, whereas the C4A isotype and a2M primarily form amide bonds with proteins (5, 6). An investigation of the binding specificities of the two human C4 isotypes has revealed that a short sequence of 4 amino acids, which lies ;100 residues C-terminal of the thioester site, accounts for the difference in binding specificity (7). Mutagenesis studies have shown that a His residue at position 1106 in C4B catalyzes the binding reaction of C4B with hydroxylbearing substrates, including water (8, 9). We have demonstrated that the mechanism of the reaction (Fig. 1) involves an attack of the His upon the thioester to form an activated acyl-imidazole
*The Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom, and †Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Received for publication January 29, 1998. Accepted for publication March 23, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work was supported by Grant MT-7081 from the Medical Research Council of Canada (to A.T.-S. and D.E.I.) and by a SOROS/FCO scholarship and a Tempus Mobility Grant (to M.G.). 2 Current address: Department of Biochemistry, Sofia University, 8 Dragan Tzankov, 1421 Sofia, Bulgaria. 3 Address correspondence and reprint requests to Dr. S. K. Alex Law, The Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K. E-mail address: alaw@ worf.molbiol.ox.ac.uk 4
Abbreviations used in this paper: a2M, a2-macroglobulin; wt, wild-type.
Copyright © 1998 by The American Association of Immunologists
intermediate; the released Cys of the thioester then acts as a base to catalyze the reaction of hydroxyl-containing substrates with the intermediate (10, 11). C4A and a2M lack a His residue at the relevant position, and their reaction with hydroxyls is not catalyzed. In this case, the thioester is relatively long-lived and is attacked directly in a noncatalytic way, thereby explaining the preference for the more nucleophilic amino groups (9). Like C4B, C3 has a His at the equivalent position (1126 in human C3, prepro-C3 numbering), reacts with hydroxyl groups, and has an activated t1/2 that is too short to be determined. However, the covalent binding of C3 to amino groups is significantly less efficient than C4B. Thus, although it is tempting to extrapolate the same chemical mechanism for C4B to account for the covalent binding activities of C3, it is necessary to obtain independent experimental evidence for C3 itself.
Materials and Methods Site directed mutagenesis, transfection, and expression Mutation of the human C3 cDNA was performed by the gapped plasmid method (12) in the vector pSVC3 (13) and inserted into the expression vector pEE6.HCMV.GS (Celltech, Slough, U.K.) (6). Stable Chinese hamster ovary K1 cell lines expressing human C3 and its variants (C3-H1126A, C3-H1126D, and C3-H1126K) were established as previously described for C4 (9, 10). The expression of C3 was assayed by an inhibition ELISA. A total of 100 ml of C3-containing tissue culture supernatant was added to 100 ml of rabbit anti-human C3 (diluted 1:60,000 in PBS, 0.1% BSA) and incubated at 37°C for 1 h. The mixture was transferred to microtiter plate wells that had been precoated with 100 ng of C3 per well (Polysorb, Nunc, Life Technologies, Paisley, U.K.). The binding of the Ab to the plate was detected using alkaline phosphatase-conjugated goat anti-rabbit IgG and was quantitated by comparison with a standard containing a known amount of C3.
Purification of proteins Using ion-exchange chromatography on a Q-Sepharose Fast Flow column (16 mm 3 200 mm) (Pharmacia, Uppsala, Sweden) equilibrated with 20 mm Tris/HCl, 50 mM e-aminocaproic acid, 5 mM EDTA, 0.1 mM Pefablock, and 50 mM NaCl (pH 7.4), expressed C3 variants were purified from the tissue culture supernatants of stable transfectants grown for 10 days after reaching confluence. The protein was eluted with a 220 ml gradient to 500 mM NaCl in the same buffer. Fractions containing C3 were loaded onto a Mono-S column (Pharmacia) (5 mm 3 50 mm) that had been equilibrated with 20 mM 2-[N-morpholino]ethanesulfonic acid and 50 mM NaCl (pH 6.0) and eluted with a 20 ml linear gradient to 500 mM NaCl. The C3-containing fractions were run on a Mono-Q column (Pharmacia) 0022-1767/98/$02.00
986
C3 REACTION MECHANISM
FIGURE 1. The covalent binding reaction of human C4B (taken from Ref. 11 with permission). using the same buffer system described for the Q-Sepharose column; elution was with a linear gradient from 50 to 500 mM NaCl over 20 ml. Plasma C3, C4, and C1s were prepared as described previously (14, 15).
Binding reactions [2-3H]glycerol (1 Ci/mmol), [2-3H]glycine (17.9 Ci/mmol), and [methyl3 H]methylamine hydrochloride (75 Ci/mmol) were obtained from Amersham (Little Chalfont, U.K.). The concentration of active protein was determined by incorporating [3H]methylamine (200 mCi/mmol) into the intact thioester bond of C3 or C4 (16). The covalent binding of C3 and C4 to [3H]glycerol (10 mM; 200 mCi/mmol) and [3H]glycine (0.1–5 mM; 200 mCi/mmol) was determined in 10 mM sodium phosphate, 140 mM NaCl, and 1 mM EDTA (pH 7.2) using human C1s (0.1% w/w) to activate C4 and trypsin (1% w/w) to activate C3; incubation was for 15 min at 37°C.
concentration of 5 mM. When the two mutant C3 molecules in which the His residue was replaced either by Ala or Asp were tested, glycerol binding was undetectable; however, close to 100% binding was observed at a glycine concentration of 5 mM. The k2/k0 values could be established using lower concentrations of glycine. In both cases, the k2/k0 values with glycine were lower than that seen with C4A but significantly higher than that observed with the plasma C3 or recombinant wt C3. Thus, replacing His1126 with either Ala or Asp converted the binding characteristics of the C3 from a hydroxyl group (glycerol) binding molecule to an amino group (glycine) binding molecule. The formation of a covalent bond in C3-H1126K
1126
C3-H
K characterization
Plasma C3 and C3-H1126K were activated with trypsin (1% w/w) in 10 mM sodium phosphate, 140 mM NaCl, and 1 mM EDTA (pH 7.2) in the presence of [2-3H]iodoacetic acid (70 mM; 145 mCi/mmol) (Amersham) for 15 min at 37°C. Cold iodoacetic acid (final concentration of 10 mM) was added, and the material was dialyzed extensively against 100 mM Tris (pH 8.0) to remove excess [3H]iodoacetic acid. The resulting material was reduced, alkylated, and digested with trypsin (2% w/w) for 16 h at 37°C. The tryptic peptides were fractionated by reverse phase HPLC on an Aquapore OD300(C18) column (Applied Biosystems, Warrington, U.K.) (100 mm 3 2 mm) in 0.2% trifluoroacetic acid over a linear gradient of acetonitrile to 60% at a flow rate of 0.2 ml/min. Peaks were collected manually, and those containing counts were sequenced on an Applied Biosystems 473A gasphase sequencer.
Results The role of His1126 in the binding reaction of C3 Three C3 variants in which His1126 was substituted with Ala, Asp, or Lys were created by site-directed mutagenesis at the cDNA level. The cDNA of the variant C3 molecules and that of wildtype (wt) C3 was introduced into the expression vector pEE6.HCMV.GS, and the plasmids were transfected into Chinese hamster ovary cells. The recombinant C3 proteins were recovered from the supernatants and were purified by conventional chromatographic techniques. The yields of active proteins were in the range of 3 to 7 mg/l of tissue culture supernatant. As judged by SDS-PAGE, the proteins were correctly processed, having the same chain structure as plasma C3. [3H]methylamine could be incorporated into the C3 a-chains, indicating the presence of an intact thioester. The binding properties of the recombinant C3 molecules with glycine and glycerol as representative small molecules with amino and hydroxyl groups, respectively, were studied and compared with C3 and C4A that had been purified from plasma (Table I). The results are expressed as k2/k0, which is the ratio of the rate constants governing transacylation to the model substrate and water, respectively. Plasma C3 and recombinant wt C3 bound to glycerol, but their reaction with glycine was very low, even at a glycine
In our previous study on C4A and C4B, we demonstrated that the residue at position 1106, if it was nucleophilic, attacks the thioester to form an intermediate. In the case of His in C4B, this intermediate was so short-lived that it was impossible to observe directly. However, a mutant in which His was replaced by Lys formed a stable amide bond during activation, and we were able to purify and characterize the cross-linked peptide (Fig. 2). Therefore, we created a C3 mutant in which His was replaced with Lys. The C3-H1126K mutant was activated with trypsin in the presence of [3H]iodoacetic acid, which labeled the free sulfhydryl group released from the thioester upon activation. After the removal of excess [3H]iodoacetic acid by dialysis, C3-H1126K was fully digested with trypsin, and the peptides were isolated by reverse phase HPLC. The peptide containing the thioester sequence was followed by radioactivity, which eluted from the column at 54% acetonitrile. Four sequences (Table II) were obtained in this fraction; peptides 1 through 3 can be identified as having originated from C3. Peptide 4 was from bovine fetuin, which was Table I. Binding of C4A, C3, and its variants to glycine and glycerol k2/k0 (M21)a Protein
Isotypic Residue
Glycerolb
Glycinec
Plasma C4A Plasma C3 Recombinant wt C3 Recombinant C3-H1126A Recombinant C3-H1126D
PCPVLD DAPVIH DAPVIH DAPVIA DAPVID
,1 16.9 33.5 ,1 ,1
4000 6 ,2 3405 666
a k2 is the second order binding rate to glycerol or glycine, and k0 is the first order hydrolysis rate. The k2/k0 values were calculated using the equation k2/k0 5 BE/ [G](1-BE), where G is the concentration of glycerol or glycine and BE is the binding efficiency, which is the fraction of thioester protein bound with glycerol or glycine, determined at the concentration of the small molecule (G). b Glycerol was used at a concentration of 10 mM in all cases. c For plasma and recombinant wt C3, the glycine concentration was 5 mM. For C4A and C3-H1126A, 0.5 mM glycine was used; for C3-H1126D, 1 mM glycine was used.
The Journal of Immunology
987
FIGURE 2. Formation of the intramolecular amide bond in C3-H1126K. Lys at position 1126 attacks the internal thioester upon activation, leading to the formation of an amide bond that is stable to subsequent trypsin digestion, peptide fractionation, and sequencing. The trypsin cleavage sites after Lys (K) and Arg (R) are marked with arrows. Note that trypsin does not cleave between Lys(K) and Pro(P), nor when the e-amino group of Lys is blocked as in the cross-linked double peptide.
most likely a contaminant from the FCS in the tissue culture medium. Peptide 1 contains the thioester sequence, but the glutamyl residue of the thioester at position 12 was undetectable. Peptide 2 Table II. Labeled tryptic peptides of C3-H1126K and C3 purified from plasma C3-H1126K
Protein a
Peptide Residue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
2
H Q L K I P V D T G P V S F G Q C* E G D E A (Q) P N V M I I (K) G Q M E T M P I V –
Plasma C3
3
4
1
S P M Y S I I T P N I L R L E S E E T M
R P T G E V Y D I E I D T L E T T – – –
H L I V T P S G C* G E E N M I G M T P V
a C3-H1126K and plasma C3 were activated in the presence of [3H]iodoacetic acid and then fully digested with trypsin. The radiolabeled peaks from reverse phase HPLC were collected and sequenced. The plasma C3 peak yielded a single sequence corresponding to the thioester peptide. A Glu (E) was detected at step 12, which is consistent with the hydrolyzed thioester. The radioactive peak from the C3-H1126K digest yielded four sequences. Peptides 1 and 2 are the thioester peptide and the specificity defining peptide which coelute in the HPLC run. The parentheses indicate that Q was not detected at step 12 and K was not detected at step 15, consistent with the postulate that these two residues are covalently linked. Peptide 3 is also from C3, which eluted from the column in the same position as the cross-linked peptides 1 and 2. Peptide 4 is from bovine fetuin (a contaminant from FCS). The [3H]-labeled Cys residue of the thioester is indicated by an asterisk.
contains Lys1126 at position 15, which was also undetected. This observation is consistent with our conjecture that Lys1126 was covalently linked to the Gln1013 of the thioester upon activation. In a similar experiment, plasma C3 was treated under identical conditions; the radioactivity associated with the thioester-containing peptide was eluted at an acetonitrile concentration of 50%, i.e., at a different position in the gradient from the C3-H1126K thioester peptide. Sequence analysis revealed that this peptide from plasma C3 had the sequence HLIVTPSGCGEENMIGMTP, where Gln1013 was identified as the residue Glu (underlined) at position 12. This sequence is to be expected when the thioester is hydrolyzed upon activation.
Discussion We have confirmed that the model that we have previously proposed for the mechanism of the binding reaction of the B isotype of human complement component C4 (10) (Fig. 1) is also applicable to C3. Upon proteolytic activation of C3 to release C3a, a conformation change occurs in the C3b fragment that allows His1126 to attack the thioester to form an acyl-imidazole intermediate with Glu1013 and release Cys1010 at the same time. The thiolate anion of Cys1010 can then act as a base to catalyze the binding of hydroxyl-bearing substrates to the intermediate. When His1126 is substituted by either Ala or Asp, no intermediate is formed, and the catalyzed reaction with hydroxyls is lost. In this situation, the direct attack on the thioester by more nucleophilic amino-bearing substrates becomes predominant, and C3 becomes C4A-like in its binding properties. When Lys is substituted for His1126, a covalent amide bond is formed between Lys1126 and Glu1013 that is resistant to further reaction. The x-ray crystal structure of a C3-C1010A derivative of the human C3d fragment has recently been determined (44), which shows that the alignment of the side chains of Ala1010 (Cys1010 in the native molecule), Gln1013, and His1126 is fully consistent with the catalyzed transacylation mechanism initially proposed for C4B (10) and demonstrated here for C3. In the model of thioester-intact
988
C3 REACTION MECHANISM Table III. Sequence comparison of the specificity-defining residues of thioester-containing proteins Proteinsa
Sequencesb
Ref.
Polymeric a-macroglobulins Human a2M Human PZP Rat a1M Rat a2M Chicken ovoM Limulus a2M
Q Q Q Q A Q
G G G G G G
(18) (19) (20) (21) (22) (17)
Monomeric a-macroglobulins Mouse MUG1 Rat a1I3 Guinea pig aM
Q K D N G C F R S S G S L F N N A M K G G Q K D S G C F R S S G S L L N N A M K G G Q K D N G C F W S S G S L L N N A I K G G
(23) (24) (25)
Mouse MUG2 Guinea pig MUG
Q K D N G C F R S S G S L F H N D I K H P Q K D N G C F R S S G T L F H N D L K G G
(23) (25)
Complement proteins Human C4A Sheep C4A
Q Q A D G S F Q D P C P V L D R S M Q G G Q R D D G S F H D P F P V M D R S M Q G G
(26) (27)
Human C4B Sheep C4B Mouse C4 Fin whale C4 Xenopus C4
Q Q Q Q Q
Q R L L K
A D G A D
D D D D T
G G G G G
S S S S A
F F F F F
Q H H H Q
D D D D E
L P P P K
S C C H V
P P P P S
V V V V V
I I I I I
H H H H H
R R R R Q
S E A G D
M M M M M
Q H Q Q L
G G G G G
G G G G G
(26) (27) (28)
Human C3 Mouse C3 Rabbit C3 Rat C3 Guinea pig C3 Cobra C3 Chicken C3 Xenopus C3 Trout C3 Lamprey C3 Hagfish C3
Q Q Q Q Q Q Q Q Q Q Q
K K K K K Q Q K Q N D
P P P P P P P P P S K
D D D D D D D D D D D
G G G G G G G G G G G
V V V V V V L L I S S
F F F F F F F F F Y F
Q Q Q Q Q K Q Q N R L
E E E E E E E E E E E
D D D D D N D N F D S
A G A G G A A A A G K
P P P P P P P P P P P
V V V V V V V V V V V
I I I I I I I I I I V
H H H H H H H H H H H
Q Q Q Q Q G K Q A R L
E E E E E E E E E E N
M M M M M M M M M M M
I I I I I L V V T Q M
G G G G G G G G G G G
G G G G G G G G N G Q
(30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40)
K K R K K R
D D E D T S
N N N N D N
G G G G G G
C C C C C C
F F F F F F
R R Q R Q R
S S Q S S K
S S S S T I
G G G G G G
S S S S I K
L L L L L L
L L L L V F
* N N N N N N
N N N N N S
A A A A A A
I I M M M L
K K K K K K
G G G G G G
c
(29)
a
PZP, pregnancy zone protein; ovoM, ovomacroglobulin or ovostatin; MUG, murinoglobulin. The position of the specificity-defining residue is indicated by an asterisk. c Unpublished work (S.K.A.L.). b
C3d calculated from the structure of the modified protein, little movement of the peptide backbone is required for thioester formation. The His1126 ring nitrogen is positioned ;4 Å from the carbonyl carbon of the thioester bond. Therefore, some local conformational change is needed to permit the nucleophilic attack required to form the acyl-imidazole intermediate. Phylogenetic analysis of the sequence data indicates that the thioester-containing complement proteins and the protease inhibitor a2M have evolved from a common ancestor (1). Most a2M sequences, including the recently published Limulus a2M (17), have an Asn at the position equivalent to His1126 of C3 (Table III) and are consequently predicted to exhibit a C4A-like binding specificity. However, the role of the covalent binding reaction in the a2M proteins is unclear. Only in the case of the monomeric a-macroglobulin, such as a1I3 of the rat, is covalent binding necessary for protease inhibitory activities. The polymeric a2M proteins rely on the physical entrapment of proteases by a Venus fly-trap-like mechanism for protease inhibition (41). a2M is an unusual protease inhibitor, in that it is active against a very wide range of proteases. This activity is possible because the protein possesses a stretch of ;30 amino acids that includes cleavage sites for proteases of all classes and with many specific-
ities. Cleavage of any bond in this area, which is called the bait region, causes a conformational change in the protein and activation of the thioester, leading to entrapment and inhibition. The physiologic roles of a2M are manyfold, but they include the clearance of both the intracellular proteases released by tissue damage and the proteases produced by invading microorganisms. The multiplicity of cleavage sites in the bait region allows proteases to be inhibited that have never before been encountered. At some time in evolution, it is possible that the ancestor of present day a2M could have become bound not to the activating bacterial proteases but also to the protease-producing organisms, and that this protein could then have acted as an opsonin. Indeed, present day a2M proteins, together with their receptors, have an opsonic function in the clearance of proteases and cytokines, as well as in the delivery of Ags to APCs (41). What then was the advantage of the mutation that led to the substitution of His for Asn and the acquisition of the catalyzed binding reaction, which has been conserved in all of the C3 proteins for which data are currently available? The answer is probably twofold: the increased efficiency of the binding to hydroxyl substrates and the increased rate of the hydrolysis of the activated thioester. Efficient binding to hydroxyl substrates has an
The Journal of Immunology obvious advantage in the immune system, since most pathogenic microorganisms are coated with a carbohydrate-rich cell wall. In addition, the thioester-binding reaction is promiscuous in the sense that, depending upon whether the catalyzed or noncatalyzed transacylation mechanism is followed, any available hydroxyl or amino group, including those on the tissues of the host, can act as acceptors. A complement component with a noncatalyzed binding reaction and, consequently, an extended t1/2 for the reactive thioester would be able to diffuse away from the site of activation and opsonize the tissues of the host. The catalyzed binding reaction ensures that binding occurs only in close proximity to the initial site of activation. It is interesting to note that in the monomeric macroglobulins, which evolved as a branch from the tetrameric macroglobulins, both Asn and His are found at the site of interest (23–25). It may not be incidental that the monomeric macroglobulins rely on covalent binding to inhibit protease activities; for the same reasons, His may have evolved as a completely distinct event from that seen in the complements (25). To date, only primates, cattle, and sheep have been shown to possess an isotype of C4 (i.e., C4A) displaying a noncatalytic covalent binding reaction (27, 42). C4 is not as potentially hazardous to the host as is C3. For classical pathway C3-convertase formation to occur, C4 must be deposited close to the activating C1, as C2 must bind to the C4 before it is in turn cleaved by C1 (43). Bound C3, on the other hand, can form an active convertase anywhere, because once factor B binds to C3, it is activated by factor D, which is present in solution and is not localized to the activating surface. A C3 with a noncatalyzed binding reaction could consequently result in considerable damage to surrounding tissues. The presence of a His residue in C3 and C4 allows the regulation of covalent bond formation, drastically reducing the t1/2 of the activated form and thus favoring the binding to hydroxyl groups in the vicinity of the C3 and C4 convertase enzymes that have been recruited to the surface of pathogenic organisms. Thus, the biologic significance of the His is to enable the functioning of both C3 and C4. In many species, the C4 gene has become duplicated, probably independently in different species (27). In some species, such as primates and bovidae, His has been replaced by Asp in one of the duplicated C4 genes and the A form of C4 has emerged. Although this replacement may facilitate C4 transacylation to hydroxyl-poor target molecules, it is noteworthy that at least one gene expressing the B form of C4 has been retained in these species, perhaps to ensure the presence of a C4 with carbohydrate-binding specificity.
Acknowledgments We thank Dr. James M. Rini of The University of Toronto for valuable comments regarding the manuscript.
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