Third component of human complement: structural analysis of the polypeptide chains of C3 and C3b

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end ofC3 mRNA. The length of C3 mRNA was determined to be 5,100 ± 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3fi chain was predicted to be Ile-Pro-MetTyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3,B chains. These data position the C3,B subunit to the NH2-terminal portion of the precursor.C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NHw-f3S-a-COOH. In additionl, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the 13 chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C31 and the NH2 terminus of the C3a subunits in pro-C3. The coding sequences of-the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.Complement is a group of interacting proteins, present in the blood of all vertebrates, that plays an essential part in the defense against microbial infections. Although the system is capable ofdirect killing and lysis ofmicroorganisms in the absence ofspecific antibodies, complement also acts as an effector ofthe humoral. immune response. The third component of complement, C3, is the central element in both the classical and the alternative pathways of complement activation. C3 contains specific sites of interaction for the classical pathway convertase C4b,2a, the alternative pathway convertase C3b,Bb, and the control proteins ,B1H and C3b inactivator. The protein is present in plasma as a two-chain molecule with disulfide-linked subunits a (Mr7 115,000) and ( (Mr) 75,000) (for review, see refs.1 and 2). C3 is synthesized as a single-chain precursor, pro-C3 (3, 4). Upon proteolytic activation by C3 convertases, C3 is cleaved into the fragments C3a (Mr, 9,000) and C3b (Mr, 180,000). C3a has anaphylactic activity and mediates inflammatory responses (5, 6). C3b has been shown to bind covalently (7) to the surfaces of foreign particles by means of a transacylation reaction (8) involving a unique structure in the C3 molecule, an internal thioester bond (9). Particle-bound C3b is cleaved by specific proteolysis into fragments C3c and C3d. The latter remains bound to the particle surface (10) and contains the thioester region (11). Clearance of C3b-coated particles by phagocytic cells involves interactions between specific cellular C3-receptor protei...

Section: Methodsmentioning

confidence: 75%

Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

Domdey¹,

Wiebauer²,

Kazmaier³

et al. 1982

“…Sequence analysis of AC-4 showed it to be contaminated with two other peptides, representing approximately 25% of the total material. We recognized the sequence of one of these as resulting from cleavage of the Asp-Pro bond at positions 5 and 6 of Bb and this 20 30 40 SDSIGASXFTGAKKCLVNLTEK…”

Section: Resultsmentioning

confidence: 99%

Partial sequence of human complement component factor B: novel type of serine protease.

Christie¹,

Gagnon²,

Porter³

1980

Factor B (a component of the alternative pathway of complement) is believed to contain the proteolytic site of the complex enzymes C3 convertase (C3bB) and C5 convertase (C3bnB). Conflicting results have been obtained in regard to the inactivation of these enzymes by diisopropyl phosphorofluoridate but it has been suggested that activated Factor B (Factor B) is a serine protease with the active site in Bb, a COOH-terminal fragment of approximately 60,000 molecular weight. Partial amino acid sequence studies of Bb derived from human Factor B have shown that the NH2-terminal 40 residues have no homology with NH-terminal sequences of other serine proteases. However, positioning of a further 170 residues out of approximately 290 residues in two continuous CNBr fragments from the COOH terminus has shown that there is a strong homology of sequence in this section. The active site residues histidine, aspartic acid, and serine all are present in positions corresponding with those of typical serine proteases. It is suggested that Factor B is a novel type of serine protease with a catalytic chain of molecular weight twice that of proteases previously studied and probably with a different activation mechanism.In the alternative pathway of activation of complement, activated Factor B (Factor B) in association with C3b forms the complex proteases C3 convertase C3bB and C5 convertase (C3b)nB (1, 2). On activation by Factor D, Factor B, a glycoprotein of about 90,000 molecular weight, is split into an NH2-terminal fragment . Ba is about 30,000 molecular weight and Bb is about 60,000 molecular weight. Medicus et al. (6) reported that C3 and C5 convertases of both pathways of activation are inhibited by diisopropyl phosphorofluoridate (iPr2P-F) and that, if a radioactive reagent is used, it is found in the Bb fragment, suggesting that Factor B is a serine protease with the active center in Bb. An earlier report, however, showed the alternative pathway CS convertase to be resistant to inhibition by iPr2P-F (7). Because the suggested catalytic peptide Bb is also about twice the length of the catalytic peptides of all other serine proteases studied so far, it appeared that Factor B could not be a typical serine protease. The amino acid sequences of many of these proteases are known and they show strong homology with each other with many highly conserved features, notably in the NH2 terminus of the catalytic peptide and in the active site residues, suggesting that all have similar catalytic and activation mechanisms (8).Sequence studies have therefore been made of Bb and have shown that, although the essential features of the active site residues of serine proteases are present and are in similar positions relative to the COOH terminus, the NH2-terminal section, which is -u300 residues longer, does not contain the characteristic NH2-terminal sequence of other serine proteases. METHODSPurification of Factor B and Fragments Ba and Bb. Minor modifications were made to the procedure of Kerr (4) for the isolation of Factor B fr...

“…C4-deficient guinea pig serum was a gift from J. P. Atkinson. C3b was prepared by treating C3 with trypsin, 1000:1 (wt/wt), for 5 min at 37°C (13). C3d was prepared by the digestion of C3 with trypsin, 200:1 (wt/wt), at pH 7.5 and 370C for 4 hr followed by separation from C3c by standard gel filtration (Sephadex G-100) and ion-exchange (DEAE-Sephacel) techniques.…”

Section: Methodsmentioning

confidence: 99%

Covalent binding and hemolytic activity of complement proteins.

Law¹,

Lichtenberg²,

Levine³

1980

166

We report the inactivation of the third component of complement (C3) In this study, we treated C3 with primary amines and compared the rate of decay of C3 hemolytic activity with the rate of loss of surface-binding activity. We find both rates to be identical, indicating that both activities are causally related. We also present data to support our hypothesis (7) that there is an internal ester bond within C3 and that C3b binds to RS by the transfer of the acyl group of the internal ester to a hydroxyl group on the RS. C4 and C5 were also treated with hydroxylamine and methylamine. C4, which binds covalently to membrane surfaces (8), behaves like C3 in these experiments. C5, which does not bind covalently to cell surfaces (8), is not inactivated by hydroxylamine and methylamine.