A B S T R A C T The failure of human serum to give rise to anaphylatoxin activity could be attributed to the presence of a potent inactivator of anaphylatoxin in human serum. The inactivator was isolated and characterized as an a-globulin with a molecular weight of approximately 310,000. It was found to abolish the activity of both anaphylatoxins, which are derived respectively from the third and the fifth component of complement, and of bradykinin. Inactivation of C3-derived anaphylatoxin and of bradykinin was accompanied by release of C-terminal arginine from these peptides. The anaphylatoxin inactivator was shown to hydrolyze the synthetic substrates hippuryl-L-arginine and hippuryl-L-lysine and to be inhibited by ethylenediaminetetraacetate (EDTA) or phenanthroline. These observations indicate that the anaphylatoxin inactivator constitues a metal-dependent enzyme resembling in specificity pancreatic carboxypeptidase B.

No abstract

The third component of human complement, C3, has been shown to be cleaved by its activating enzyme C3 convertase (1) and by the C3 inactivator complex (2) into two fragments called C3a and C3b (3, 1). The C3a fragment which is the smaller of the two pieces was found to have anaphylatoxin activity (3, 4). Streptokinase-activated plasminogen also was found to cleave a small fragment off the C3 molecule which, however, was different from C3a in that it exhibited leukocyte chemotactic activity, but no anaphylatoxin activity (5, 6).The present study was initiated (a) to investigate cleavage of the native C3 molecule by a proteolytic enzyme with defined and restricted bond specificity, such as trypsin; (b) to isolate and characterize the fragments obtained; and, if possible, (c) to relate biological activities to defined fragments of the C3 molecule. It was found that trypsin cleaves the C3 molecule into at least four distinct fragments, two of which closely resemble C3a and C3b, and that both the anaphylatoxin and the chemotactic activities are resident in the C3a, although in different regions of this fragment. The C3a fragment could be isolated in amounts sufficient for physical and chemical characterization, and an antiserum capable of inhibiting its biological activity could be produced. In the course of this work, an inactivator of anaphylatoxin was found in human serum fractions and evidence was obtained, suggesting that it destroys anaphylatoxin activity by an enzymatic mechanism. Part of the results contained in this communication were presented earlier in abridged form (7,8).* This is publication number 317 from

Participation of complement in the pathogenesis of a variety of immunologic diseases has been established . Consequently, immune complexes and other complement activating substances have been searched for in serum and other body fluids of respective patients . Agnello, Winchester, and Kunkel have utilized isolated Clq for the detection of Clq-precipitable substances in pathological sera and joint fluids (1, 2) . In pursuing the approach of these authors it was possible to develop a procedure which is considerably more sensitive than the Clq precipitin method.This procedure is based on the inhibition by immune complexes of radiolabeled Clq uptake to sensitized erythrocytes . The degree of inhibition can be accurately quantitated. In the following we describe the development of the Clq deviation test, its sensitivity with respect to different Clq reactants, and its application to the exploration of the Clq-reactive material in pathological sera. Materials and MethodsClq. Highly purified Clq was isolated from fresh human serum as previously described (3). The isolated protein was stored at a concentration of 1 mg/ml in 0.1 M phosphate buffer, pH 7, at -70°C until used .Clq was labeled with '251 according to the method of McConahey and Dixon (4) . Specifically, 0.6 mCi of carrier free '251 (ICN Laboratories, Irvine, Calif.) and 10 pg of chloramine T were added to 1 mg of Clq. The mixture was stirred for 10 min at 4°C and the reaction was stopped by addition of 10 j ug of sodium metabisulfite. The labeled protein was dialyzed three times against 10 liters of 0.1 M phosphate buffer, pH 7.2, for 36 h at 4°C and then subjected to ultracentrifugation in a Spinco no . 40 rotor (Beckman Instruments, Inc., Spinco Div., Palo Alto, Calif.) at 35,000 rpm and 4°C for 60 min. Uptake of 1251 was approximately 50%, the specific radioactivity ranged between 0.25 and 0.4 ACi/ Ag protein. Bovine serum albumin (BSA)' at a concentration of 1 mg/ml was added before storage at

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...

This study describes the presence of a receptor for fluid phase human C3 and C3b on Raji cell membranes. The binding of C3 and C3b was demonstrated indirectly by a fluoresceinated anti-C3 serum and directly by using radioiodinated proteins. No other complement proteins or serum factors were needed to mediate binding of C3 and C3b to the receptor. The possibility of enzymatic cleavage of C3 before or after its attachment on the cell membrane was ruled out by the demonstration of antigenically intact C3 on Raji cells. Inhibition and dissociation of Raji cell-EAC1423 rosettes by C3 and C3b indicated that both of these proteins bind to the same receptor site or closely associated receptor sites on Raji cells. C3b-bearing Raji cells were immune adherence negative, indicating that C3b binding to the receptor is brought about through the immune adherence region of the molecule and not the C3d portion. The C3 receptor on Raji cell membranes is uniformly distributed and can move on the membrane plane. Approximately 4 x 105 molecules of C3 or C3b bind per Raji cell. The receptor had a higher affinity for C3 than C3b, as was shown by uptake experiments and inhibition of Raji cell-EAC1423 rosette formation. Apart from the described receptor for C3 and C3b another specific receptor for C3b inactivator-cleaved C3b (C3d) bound to red cells was shown to be present on Raji cells. Raji cells cultured in medium containing fresh normal human serum and cobra venom factor were lysed. Similar results were obtained when C3b-bearing Raji cells were cultured in medium with fresh normal human serum. The lytic effect could be abolished by inactivating serum C3 proactivator (C3PA) and required C6. It was concluded that C3b bound to the Raji cell membrane activates the complement system through the alternate pathway and results in membrane damage and cytolysis. It is postulated that cell destruction by this mechanism may play an important role in vivo in controlling cell growth.

The occurrence of 19S anti-IgG has been frequently observed during hyperimmunization of rabbits with various antigens including bacteria (1, 2), soluble proteins (3), autologous IgG (4), and Trypanosoma (5). There are only a few reports which mention the occurrence of 7S anti-IgG. Williams and Kunkel (6), for example, described both 7S anti-IgG and 19S anti-IgG in some rabbits immunized with autologous IgG.Rabbit streptococcal group-specific antisera provided a unique opportunity to reexamine the nature of the 19S and 7S anti-IgGs because they occur commonly during intravenous hyperimmunization with whole streptococcal vaccines. From preliminary studies in collaboration with Dr. Charles Christian, it was learned that concentrations of 19S anti-IgG were much greater in streptococcal antisera than those in antisera from rabbits immunized with Gram-negative organisms. Furthermore, it has been observed that occasional hyperimmune antistreptococcal sera were viscous, and in some instances, a gel or cryoprecipitate formed in the cold. Analytical ultracentrifugation of these sera revealed the presence of intermediate complexes which were indicative of 7S anti-IgG. One cryoglobulin was described in an earlier report (7), but the anti-IgG activity was not observed at the time.The antisera from 88 rabbits hyperimmunized with Groups A and C streptococci have been examined for the occurrence of 19S and 7S anti-IgGs. These antibodies are much less common and occur in lower concentrations in the antisera of rabbits hyperimmunized with pneumococci, than in streptococcal sera. This finding suggests a specific role for antigen in the induction of antiIgGs. Studies on inbred rabbit families suggest further that the ability to produce 7S anti-IgG after immunization with streptococci may be an inherited genetic trait.

Human bone marrow-derived (B type) lymphocytes have receptors for