The purification and characterization of subcomponent C1s of the first component of bovine complement.

Bovine C1s, a subcomponent of the first component of complement, was purified in good yield by a combination of euglobulin precipitation and ion-exchange and molecular-sieve chromatography. Approx. 10 mg can be obtained from 3 litres of serum, representing a yield of 11%. The C1s is obtained in zymogen form, with a mol.wt. of 85000-88000, determined by gel filtration and SDS/polyacrylamide-gel electrophoresis. It is haemolytically active when tested with human C1q and C1r. Activation can be achieved by incubation with human C1r, resulting in cleavage of the C1s chain into two chains of 65000 and 27000 mol.wt. and the generation of an isoleucine N-terminal residue on the smaller chain. Active C1s binds an equimolar amount of di-isopropyl phosphorfluoridate to the smaller chain, which is the C-terminal part in the zymogen. The chains can be separated by ion-exchange in 8 M-urea. All of these characteristics show that bovine C1s is very similar to its human counterpart.     

Subunit composition and structure of subcomponent C1q of the first component of human complement.

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.     

Purification and characterization of subcomponent C1q of the first component of bovine complement

Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.     

The purification and properties of the second component of human complement.

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.     

Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.     

Chemical studies on the isolated collagen-like and globular fragment of complement component C1q. Comparative studies on bovine and human C1q

Both the collagen-like and the globular fragments of a subcomponent C1q of the first component of bovine and human complement were highly purified by enzymic digestion followed by gel filtration. Analyses by polyacrylamide gel electrophoresis showed that the former was composed of covalently linked peptide chains with an average molecular weight of 14 000, and that the latter was composed of three non-covalently linked peptide chains each having a molecular weight of approximately 15 000. Great similarities between amino acid compositions of the globular fragments and some similarities between those of the collagen-like fragments were found. Moreover, great similarities of amino acid compositions were found among three non-covalently linked chains of each globular fragment as well as between the corresponding chains of both globular fragments. These results suggested that both the collagen-like and the globular domains on the C1q molecule remained highly conserved in its evolution.     

The isolation and structure of C4, the fourth component of human complement.

The fourth component of complement, C4, was isolated from human serum in good yield, and in confirmation of previous reports was shown to be formed from three peptide chains, alpha, beta and gamma, with apparent mol.wts. 90 000, 80 000 and 30 000 respectively. Preparative methods are described for the isolation of the three peptide chains and their amino acid analyses reported. Component C4 contains 7.0% carbohydrate, alpha-chain 8.6% and the beta-chain 5.6%. The N-terminal amino acid sequences are given for 12 residues of the alpha-chain, eight of the beta-chain and 19 of the gamma-chain.     

Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with alpha 2-macroglobulin.

The alpha polypeptide chain of the complement protein C3 splits into two fragments of 74 000 and 46 000 apparent mol.wt. under certain conditions used to prepare the protein for SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. The cleavage reaction occurs over a wide range of temperatures and from pH 4.6 to 10.6 in the presence of denaturants such as urea, SDS and guanidine hydrochloride. It is also induced by heat-denaturation of C3 in the absence of chemical denaturants. The reaction occurs only with haemolytically active C3, and is not observed with hydroxylamine-inactivated C3 or with C3b. A similar cleavage of the alpha-chain of complement component C4 occurs under the same conditions, forming fragments of 53 000 and 41 000 apparent mol.wt. This reaction is again specific for haemolytically active C4, and does not occur with C4b or hydroxylamine-inactivated C4. The complement component C5, although structurally similar to C3 and C4, does not undergo a reaction of this type. The characteristics of the denaturation-induced cleavage of C3 and C4 match those described for the 'heat-induced' cleavage of alpha 2-macroglobulin [Harpel, Hayes & Hugli (1979) J. Biol. Chem. 254, 8669-8678]. Cleavage of alpha 2-macroglobulin is also specific for the active form of the protein, and does not occur with chemically inactivated or proteinase-cleaved forms. The unusual conditions and specificity of the peptide-bond cleavage in all three proteins suggest that it is an autolytic process rather than being the result of trace proteinase contamination. The active forms of C3, C4 and alpha 2-macroglobulin have the transient ability to form covalent bonds after activation. The autolytic cleavage reaction is likely to be related to the covalent-bond-forming reactions of these proteins.     

Isolation and characterization of C1q, a subcomponent of the first component of complement, from human and rabbit sera

1. C1q, a subcomponent of the first component of complement, has been isolated, in a haemolytically active and soluble form, by ion-exchange chromatography and gel filtration, from human and rabbit sera. Yields ranged from 10 to 25mg/litre of serum and the activity of final preparations was consistently in the range 5x10(3)-15x10(3) C1qH(50) units/mg. 2. The molecular weights of human and rabbit subcomponent C1q were 409600 and 417600, as determined by sedimentation equilibrium studies. 3. Subcomponent C1q from both species was shown to be composed of non-covalently linked subunits of approximately 57000 molecular weight as determined by gel-filtration or sedimentation equilibrium studies in 5.3m-guanidinium chloride. Reduction or oxidation of human and rabbit subcomponent C1q yielded three chains each having a molecular weight of approximately 23000 and which differed slightly in amino acid composition but markedly in carbohydrate content. The oxidized chains were separated, on a preparative scale, by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 4. Both human and rabbit subcomponent C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 8% carbohydrate. Glutamic acid and aspartic acid were the free N-terminal amino acids of human subcomponent C1q whereas only serine was found in rabbit subcomponent C1q. 5. Collagenase digestion of human or rabbit subcomponent C1q caused a rapid loss of haemolytic activity which correlated with the breakdown of collagenous regions in the molecule.     

Purification and structural analysis of the fourth component of human complement.

The fourth component of human complement (C4) has been purified in 20% yield from fresh plasma using as starting material the 5-12% poly(ethylene glycol) precipitate which had been depleted of plasminogen by an affinity adsorbent. Sequential ion-exchange chromatography on diethylaminoethylcellulose, QAE-Sephadex, and DEAE-Bio-Gel A resulted in C4 homogeneous by immunological criteria and by polyacrylamide gel electrophoresis, the last chromatographic step achieving separation of native from inactivated C4. Reduction with 20 mM dithiothreitol for 2 h at 37 degrees C in 0.25 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride, pH 8.6, effected cleavage of the interchain disulfide bonds. A three-chain structure for C4 was confirmed, and molecular weight estimates of 93 000 +/- 9300, 75 000 +/- 7500, and 30 000 +/- 3000 determined for the alpha, beta, and gamma chains, respectively. The effects of known inactivators of C4 upon the chains of C4 were investigated, confirming that the inactivations by C1s and trypsin were accompanied by the fragmentation of the alpha chain. Inactivation of C4 by hydrazine, on the other hand, produced no detectable change in chain size. Separation of the chains was accomplished by gel filtration in the presence of 1 M acetic acid. Amino acid compositions of native C4 and the constitutive chains have been performed, and N-terminal sequences of the latter established by automated Edman degradation.     

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.     

Purification and characterization of subcomponent C1q of the first component of mouse complement.

1. Mouse C1q, a subcomponent of the first component of complement, has been purified in a highly haemolytically active form by a combination of precipitation with EGTA, ion-exchange chromatography and gel filtration. Yields ranged from 3 to 5 mg/200 ml of serum, and the activity of final preparations was in the range of 2 X 10(13)-4 X 10(13) C1q effective molecules/mg. 2. The molecular weight of mouse C1q was 439 500 +/- 1586, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. Mouse C1q was shown to be composed of non-covalently linked subunits, all being in the molecular-weight range 45 000-46 000, and three covalently linked chains each having a molecular weight of approx. 23 000 as determined on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate by using non-covalently and covalently linked subunits of human C1q as markers with known molecular weights calculated theoretically previously [Porter & Reid (1978) Nature (London) 275, 699-704]. 4. Mouse C1q contained hydroxyproline, hydroxylysine, a high percentage of glycine and approx. 9% carbohydrate. The absorption coefficient and nitrogen content of C1q were also determined.     

Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement.

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Physicochemical and immunochemical studies on bovine IgA and glycoprotein-a

1. Bovine secretory IgA (SIgA) from colostrum (mol. wt. about 410,000) is composed of four alpha-chains (mol. wt. 61,000), four light chains (mol. wt. 23,000) and one molecule of glycoprotein-a (mol. wt. 70,000-86,000). The alpha-chains are antigenically and physicochemically distinct from the heavy chains of IgM, IgG1 and IgG2 while the light chains are identical to those occurring on other bovine immunoglobulins. Glycoprotein-a and bovine free secretory component are identical and the former name should be abolished. Much of this protein is covalently bonded to IgA. 2. The gel filtration behavior of serum IgA suggests it is a dimer. 3. The elution behavior of IgA and SIgA from ion-exchange columns and the solubility characteristic of SIgA in the presence of Zn2+ are similar to those of human and rabbit IgA. 4. The disc electrophoretic behavior of IgA and SIgA are distinct from IgM, dimeric IgG1, 7-S IgG and glycoprotein-a. Dimeric IgG1 (s20,w = 9.5) is abundant in colostrum and is similar in size to SIgA. 5. Bovine IgA shows physicochemical and immunochemical heterogeneity when studied by gel filtration, disc electrophoresis, immunoelectrophoresis and ultracentrifugational analyses. Lacrimal and nasal SIgA possess antigenic determinants absent on colostral SIgA.     

The structure and enzymic activities of the C1r and C1s subcomponents of C1, the first component of human serum complement.

The subcomponents C1r and C1s and their activated forms C-1r and C-1s were each found to have mol.wts. in dissociating solvents of about 83000. The amino acid compositions of each were similar, but there were significant differences in the monosaccharide analyses of subcomponents C1r and C1s, whether activated or not. Subcomponents C1r and C1s have only one polypeptide chain, but subcomponents C-1r and C-1s each contain two peptide chains of approx. mol.wts. 56000 ("a" chain) and 27000 ("b" chain). The amino acid analyses of the "a" chains from each activated subcomponent are similar, as are those of the "b" chains. The N-terminal amino acid sequence of 29 residues of the C-1s "a" chain was determined, but the C-1r "a" chain has blocked N-terminal amino acid. The 20 N-terminal residues of both "b" chains are similar, but not identical, and both show obvious homology with other serine proteinases. The difference in polysaccharide content of the subcomponents C-1r and C-1s is most marked in the 'b' chains. When tested on synthetic amino acid esters, subcomponent C-1r hydrolysed both lysine and tyrosine ester bonds, but subcomponent C-1r did not hydrolyse any amino acid esters tested nor any protein substrate except subcomponent C1s. The lysine esterase activity of subcomponent C1s provides a rapid and sensitive assay of the subcomponent.     

Trypanosoma lewisi: restriction of alternative complement pathway C3/C5 convertase activity

The rat parasite Trypanosoma lewisi was incubated in vitro with rat or human serum, washed, and extracted in detergent. Extracts were fractionated by electrophoresis in denaturing gels, transferred to nitrocellulose, allowed to renature, then immunoblotted with polyclonal antibodies to rat complement component C3 and human complement components C3, C5, and factor B. Molecules that reacted with these antibodies were detected in the extracts. Fragments of rat C3 were detected in extracts of parasites that had not been exposed to serum in vitro. Additional complement deposition occurred during in vitro incubations; human complement components deposited in vitro could be distinguished from rat components deposited in vivo. Complement deposition in vitro required magnesium ions and did not occur when heat inactivated serum was used. Components reacting with antibodies to human C3 included a group of bands with molecular weights higher than C3 alpha or beta chains. Blotting with affinity purified, chain specific antibodies demonstrated that a 68 kDa component on parasites is C3 beta and that a 44 kDa molecule is derived from C3 alpha. A 73 kDa component that was difficult to resolve from C3 beta is probably also a C3 alpha fragment. This suggests that an inactive iC3b-like molecule is present on parasites. Kinetic studies showed that cleavage of C3 alpha is rapid and that the amount of C3 alpha fragments and C3 beta on intact parasites reached a steady state after 15 min. When parasites were trypsinized prior to incubation in C5 or C6 deficient serum, the rate and extent of C3 and C5 deposition increased. Unprocessed C3 alpha' and C5 alpha' chains were detected. Trypsinized parasites were lysed by the alternative complement pathway in normal serum. Intact parasites could be lysed by complement in the presence of antibody. The data support our previous suggestion that trypsin sensitive surface proteins on intact T. lewisi limit alternative pathway activity by restricting C3/C5 convertase activity.     

Isolation of the globular region of the subcomponent q of the C1 component of complement.

Digestion after heat treatment of the subcomponent q of the C1 component of complement by collagenase leads to the isolation of the globular region of the protein. This product ('heads') is composed of three chains giving an overall molecular weight of about 57000. About half of the collagen-like region present in C1 q is lost after digestion. The 'heads' are shown to be soluble and hemolytically active products.     

Distribution of complement protein C4 concentrations in bovine plasma

Complement component C4 concentrations were measured in 40 pure bred Hereford cattle and 40 cattle from a mixed breed herd. Significant differences were not observed between the two groups studied nor between bulls and cows. However, the distribution of C4 concentrations was relatively disperse and appeared polymodal suggesting the presence of two isotypes of C4. Polyacrylamide gel electrophoresis of immunoprecipitated bovine C4 showed many samples to have two C4 alpha chains differing in relative molecular mass by about 1800. Isoelectric focusing of bovine plasma in agarose gels followed by immunofixation with specific anti-C4 antisera revealed two populations of native C4 differing in pI by about 0.3 pH unit. An association between the type of C4 alpha chain present and the pI of the native C4 molecule was observed. Collectively these findings indicate the presence of two structural C4 genetic loci in cattle.     

The structure of bovine complement component 3 reveals the basis for thioester function

The third component of complement (C3) is a 190 kDa glycoprotein essential for eliciting the complement response. The protein consists of two polypeptide chains (alpha and beta) held together with a single disulfide bridge. The beta-chain is composed of six MG domains, one of which is shared with the alpha-chain. The disulfide bridge connecting the chains is positioned in the shared MG domain. The alpha-chain consists of the anaphylatoxin domain, three MG domains, a CUB domain, an alpha(6)/alpha(6)-barrel domain and the C-terminal C345c domain. An internal thioester in the alpha-chain of C3 (present in C4 but not in C5) is cleaved during complement activation. This mediates covalent attachment of the activated C3b to immune complexes and invading microorganisms, thereby opsonizing the target. We present the structure of bovine C3 determined at 3 Angstroms resolution. The structure shows that the ester is buried deeply between the thioester domain and the properdin binding domain, in agreement with the human structure. This domain interface is broken upon activation, allowing nucleophile access. The structure of bovine C3 clearly demonstrates that the main chain around the thioester undergoes a helical transition upon activation. This rearrangement is proposed to be the basis for the high level of reactivity of the thioester group. A strictly conserved glutamate residue is suggested to function catalytically in thioester proteins. Structure-based design of inhibitors of C3 activation may target a conserved pocket between the alpha-chain and the beta-chain of C3, which appears essential for conformational changes in C3.