C5 Convertase of the Alternative Pathway of Complement

Abstract

Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (k cat ). However, the surface-bound enzyme, ZymC3b,Bb had a K m (1.4 M) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (K m ‫؍‬ 24 M). The k cat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s ؊1 and that for soluble C3b,Bb was 0.0110 s ؊1 . Both forms of the enzyme had a low turnover number at V max (0.23 to 0.68 C5/min/enzyme). Substituting Mg 2؉ for Ni 2؉ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the K m determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.C5 convertases are serine proteases that cleave C5, the fifth component of complement. The cleavage of C5 is the last enzymatic step in the complement activation cascade resulting in the formation of two biologically important fragments, C5a and C5b.1 Both fragments play vital roles in killing microorganisms. C5a, the smaller fragment, is a potent chemotactic and spasmogenic anaphylatoxin. It mediates inflammatory responses by stimulating neutrophils and phagocytes. C5b, the larger fragment, initiates the formation of the membrane attack complex (C5b-9) which results in the lysis of bacteria and other pathogens. Although complement has important functions in host defense, it also contributes to the pathology in many inflammatory diseases (1), in xenotransplant rejection (2), and in reperfusion injury (3). This has prompted a search for inhibitors that can control activation of C5. Specific inhibition of C5 activation would preserve the immune clearance and opsonization functions of complement which depend on C3b, but it would prevent the generation of both C5a and C5b-9. Recent approaches in blocking complement activation include inhibitory anti-C5 antibodies (4), synthetic peptides (5, 6), synthetic protease inhibitors (7,8), soluble constructs of ...

Activation of Complement Component C5

“…The cells were washed 5 times with GVB ϩϩ to remove unbound C2 and C3. The number of C3b deposited per E Man was determined by measuring the number of radiolabeled factor B molecules bound as Bb, the activated product of factor B, to E Man M1,C3bC4b cells as described (51). The procedure was repeated until the desired numbers of C3b/ E Man were obtained.…”

“…The cells were washed five times with GVB ϩϩ to remove C3 and unbound C2. The total number of radiolabeled C3b molecules bound to ZymM1,C3bC4b was determined by using [ 125 I]C3 (50) as described for alternative pathway C5 convertases (51,52). ZymM1,C3bC4b cells were washed and the procedure repeated until the desired numbers of C3b/Zym were obtained.…”

“…Quantitation of Reaction Products-The sensitivity of chicken erythrocytes (E C ) to human C5b,6 was employed to measure the amount of C5b,6 formed in hemolytic assays as described (31,51). Controls with C5 and C6 but no C2 were subtracted as background.…”

“…After 10-min incubation at 37°C, further cleavage of C5 was prevented by transferring assay tubes to an ice bath and adding 225 l of ice-cold GVBE. Appropriately diluted assay mixtures were titrated for C5b,6 formation by hemolytic assays using E C , and the amount of C5b,6 formed was quantitated from standard curves generated with purified C5b,6 as described (31,51).…”

“…To ensure that C5b,6 was not formed from the C5 and C6 in the pooled normal human serum used as a source of C7-9 during the lysis of E C , reaction mixtures containing C5 convertase but no purified C5 or C6 were used as controls. C5b,6 concentration was quantitated from a standard curve using purified C5b,6 as described previously (51).…”