Third component of complement (C3): structural properties in relation to functions.

Abstract

The third component of complement (C3) fulfills a pivotal role in the functions of the complement system. We have investigated the topological relationships among its polypeptide chains, physiologic fragments, enzyme attack regions, and functional sites. C3 consists of two chains (a and ,6) which are linked by disulfide bonds and noncovalent forces and which have molecular weights of, respectively, 120,000 and 75,000. C3 is activated by action of C3 convertase on the a-chain. With hydrolysis of one polypeptide bond, C3a, the 9000 dalton activation peptide is dislocated from the NH2-terminal portion of the a-chain. A previously concealed binding region is thereby transiently revealed in the C3b-fragment (181,000 dalton) which displays affinity for apparently nonspecific acceptors present on biological membranes. Binding of nascent C3b to membranes occurs through the C3d portion of the fragment because subsequent action of the C3b-inactivator or trypsin on bound C3b causes release of C3c, but not of C3d. Bound C3b and C3d possess stable sites that are capable of binding to specific receptors present on a limited variety of cells. We propose that all known physiologically occurring fragments of C3 arise by enzymatic cleavage of the a-chain: C3a, C3b, C3c, and C3d. Whereas C3a (a,) and C3d (a2) consist of a single chain and C3b consists of two chains (a' and ,B), C3c is composed of the entire ,3-chain and multiple fragments of the a-chain, each of which is linked by disulfide bonds to the ,3-chain.The aim of this communication is to describe the topological relationships among chains, fragments, functional sites, and enzymatic attack regions of the C3 molecule (third component of complement).C3, which was first recognized and isolated in 1960 (1), fulfills multiple physiological functions in host defense against pathogenic microorganisms and probably transformed host cells. It occurs in plasma and in other body fluids in inactive, but activatable form. The activating enzyme, C3 convertase or C4,2, cleaves the molecule into two fragments, C3a and C3b (2, 3). The activation peptide, C3a, constitutes one of the two known anaphylatoxins. In very low concentrations the peptide effects release of histamine from mast cells (4), chemotactic migration of polymorphonuclear leukocytes (3, 5), and contraction of smooth muscle (2,3,6). In vivo it is phlogogenic and causes formation of cutaneous edema and erythema (7,8).The large fragment, C3b, in its nascent state, is capable of binding to the surface of cells (9) For this purpose fresh human serum containing 0.05% NaN3 was incubated for 5 days at 370, under which conditions C3 is degraded and these fragments accumulate. Alternatively, 250 ml of fresh human serum was incubated at 370 for 1 hr with 2 mg of isolated cobra venom factor (26) and was subsequently held at 40 overnight. C3c and C3d were isolated by a threestep procedure. Treated serum (250 ml) was dialyzed against phosphate buffer, pH 8.1, having a conductance of 4 mmho/ cm. The serum was applied to a 3-lit...

Biosynthesis of the third component of complement (C3) in vitro by monocytes from both normal and homozygous C3-deficient humans.

“…The radiolabeled C3 protein produced in vitro had the approximate molecular weight and subunit structure of purified human serum C3. The estimates of molecular size of intact C3 (both the purified serum C3 and that produced in vitro) were somewhat different from that (190,000) reported in the literature (23). This difference was not observed in analysis of the a-and ,3-subunits, the sizes of which corresponded closely to published figures (a-chain 120,000; /-chain 75,000) (23).…”

“…The estimates of molecular size of intact C3 (both the purified serum C3 and that produced in vitro) were somewhat different from that (190,000) reported in the literature (23). This difference was not observed in analysis of the a-and ,3-subunits, the sizes of which corresponded closely to published figures (a-chain 120,000; /-chain 75,000) (23). Since the size ofintact C3 exceeded that of the largest marker protein, a precise estimate of its molecular weight could not be determined under nonreducing conditions.…”

Identification and structural characterization of two incompletely processed forms of the fourth component of human complement.

“…The Mr of a2M and the a-chain of C3 were -185,000 (25) and -110,000 (26), respectively, and were distinct from p168 and p125 (data not shown). Immunoprecipitation of a2M, C3, and C4 from a human hepatoma cell line known to synthesize and secrete these three molecules (27) also revealed that p168 and p125 are not C3 or a2M (see below).…”

Human complement C3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution.

“…From previous studies it was concluded that proteolytic cleavage of the a'-chain of C3b (36,37) by C3bINA splits the molecule into the immunochemically defined fragments C3c and C3d (38). Our results indicate that in complete absence of/31H, C3bINA does not cleave C3b in solution (Fig.…”