Mass-spectrometry assisted heavy-atom derivative screening of human Fc gamma RIII crystals.

A heavy-atom screening method aided by mass spectrometry is described here. Using mass spectrometry, several heavy-atom compounds have been screened in order to obtain potential phasing derivatives for the crystals of a human immunoglobulin Fc receptor, Fc gamma RIII. Of these, HgCl(2), trimethyllead acetate (TMLA), KAu(CN)(2), K(2)PtCl(4) and PbAc(2) reacted with Fc gamma RIII in solution, whereas KAuCl(4), ethylmercuric thiosalicylate (EMTS) and Na(2)WO(4) did not. To validate the mass-spectrometry results, these heavy-atom compounds were also used to soak crystals of Fc gamma RIII and crystallographic data were collected after soaking. The calculated R(iso) indicated that HgCl(2), TMLA, K(2)PtCl(4) and PbAc(2) were likely to form derivatives, whereas KAu(CN)(2) and Na(2)WO(4) were not. The anomalous difference Patterson maps calculated for the HgCl(2) and TMLA derivative data sets were of good quality and can readily be interpreted by hand. In general, the number of binding sites obtained from the crystallographic phase refinement of the derivatives agrees with those obtained from the mass spectrometry, suggesting that mass spectrometry can be applied for rapid searching of suitable heavy-atom derivatives for X-ray crystallography.

C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway.

Mast cells are known to accumulate at sites of inflammation, however, the chemotaxins involved are undefined. Since most natural leukocyte secretagogues also induce cell migration, and since the anaphylatoxins C3a and C5a are mast cell secretagogues, we hypothesized that both C3a and C5a are also mast cell chemotaxins. Here we report that C3a and C5a are, in fact, potent chemotaxins for the human mast cell line HMC-1. The optimal concentrations, half-maximal effective concentrations (a measure of agonist potency) and the efficacy (response at the optimal concentration) compared with medium control were, for C3a: 10 nM, 0.5 nM, and 256%, respectively; for C5a: 1 nM, 10 pM and 145%. Chemotaxis of HMC-1 cells to both C3a and C5a was blocked by pertussis toxin, suggesting that Gi-coupled receptors are involved in signal transduction. C3a and C5a also induced transient pertussis toxin-inhibitable increases in [Ca2+]i (ED50 = 1 nM for both) that could be homologously but not heterologously desensitized, suggesting that the receptors for C3a and C5a are distinct. These results make C3a the most effective mast cell chemotaxin identified to date. The chemotactic potency described here for C3a is also 100- to 1000-fold greater than for all of its previously described cellular actions. Direct chemoattraction of mast cells by C3a and C5a may help explain the rapid accumulation of mast cells at sites of inflammation.

Production and characterization of the monoclonal antibody against SGP120, a novel serum protein.

A monoclonal antibody designated B1.9.E-2 was produced and characterized to facilitate study of the immunizing antigen-a serum glycoprotein of 120 kDa (sgp 120) of unknown function. The antibody, produced in mice, reacts with 1-2 epitopes located in the N-terminal region of the sgp 120 molecule. The affinity of the reaction, as determined by Scatchard analysis, is Ka = 1.13 x 10(10) I/M and is of a hydrophobic nature. The monoclonal antibody can be purified to a high degree by a modified caprylic acid method and by protein G FPLC chromatography column. B1.9.E-2 does not trigger the complement cascade, as determined by C3 RIA and immune complex solubilization assays. Both affinity purified (C4b Sepharose) and chromatographically isolated (using jacqualine agarose) sgp 120 were recognized by B1.9.E-2. The monoclonal antibody can be utilized for affinity purification of sgp 120, for detection of intact or cleaved sgp 120 in biological samples of healthy and ill individuals, and for the number of functional and neutralization assays aimed at resolving the physiologic role of sgp 120.

Further identification of human plasma glycoproteins interacting with the galactose-specific lectin Jacalin.

In this report we show that Jacalin binds the heme-binding protein hemopexin and the C4b-binding protein sgp120 in human plasma. The interaction of Jacalin with hemopexin confirms that a single O-linked oligosaccharide is sufficient to mediate binding of a protein to this lectin. Retention of sgp120 by immobilized Jacalin demonstrated that this protein was O-glycosylated and, therefore, clearly different from another C4b-binding protein, the complement protein C2 which is physicochemically similar but exclusively N-glycosylated. In addition, Jacalin was also shown to bind several proteolytic enzymes which remain to be identified.

TnphoA-mediated disruption of K54 capsular polysaccharide genes in Escherichia coli confers serum sensitivity.

To assess whether non-K1, group 2 capsular serotypes are important in conferring serum resistance to extraintestinal isolates of Escherichia coli, a K54 blood isolate (CP9) was evaluated as a model pathogen. Transposon mutagenesis (TnphoA) was used to generate isogenic capsule-negative mutants. CP9 was resistant to the bactericidal effects of serum, growing in 80% serum. In contrast, all of the capsule-negative mutants had an increased sensitivity to 80% normal human serum, undergoing a 2- to 3-log kill over 3 h when starting inocula of 10(4) to 10(7) CFU/ml were used. The killing of the capsule-negative strains was mediated through the alternative complement pathway and not by lysozyme or beta-lysins. The protective effect of the K54 capsule against the bactericidal activity of serum was not through inhibition of the complement cascade, nor did it appear to be through a difference in the binding of C3.

Potassium cyanide protects Escherichia coli from complement killing by the inhibition of C3 convertase activity.

The exact mechanism by which deposited C5b-9 complexes kill Gram-negative bacteria is unclear. It has been proposed that during complement activation the membrane attack complex triggers an energy dependent process in Gram-negative bacteria that mediates destruction of the inner membrane. This observation in part resulted from the survival of Gram-negative bacteria that were incubated with an uncoupler (DNP) or an inhibitor (KCN) of oxidative phosphorylation during complement activation. In a reexamination of this issue we employed potassium cyanide (KCN) to block energy dependent pathways and observed a dose dependent inhibition of C9 uptake on E. coli J5 during serum incubation, suggesting that cyanide was interfering with complement activation. To verify the effect on complement activation we chose specifically to study the effects of KCN on the C3 convertase of the classical pathway. Sensitized sheep erythrocytes were employed as our model system. This system allowed us to construct a series of stable intermediates that were used to test the effect of cyanide on the formation and activity of precursors of the classical pathway C3 convertase. The data illustrate that the concentrations of potassium cyanide that inhibit complement killing of J5 also inhibit C3 convertase activity on sensitized sheep erythrocytes. The results of this study refute the principal observation made by other investigators, that potassium cyanide protects bacteria from complement killing by inhibiting bacterial energy dependent pathways that spark inner membrane destruction. A better scenario is that the organisms survive because cyanide inhibits complement activation.

Regulation of complement activity by vaccinia virus complement-control protein.

A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.

A rapid FPLC method for purification of the third component of human and guinea pig complement.

A method is described for the purification of human and guinea pig C3 from small amounts of serum. This procedure requires only two steps--polyethylene glycol (PEG) precipitation and fast protein liquid chromatography (FPLC) Mono Q HR 10/10 ion exchange chromatography. The protocol takes less than two hours to complete and yields 4-6 mg of purified C3. Similar results, in terms of antigenic and functional recovery, were obtained for both human and guinea pig components. About 67% of C3 antigen was recovered from eluted fractions with fully preserved specific activity. Isolated C3 was over 95% pure as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography; this level of purity was confirmed by the absence of any observable contamination as assessed by immunoelectrophoresis using high titer anti-whole human serum. This method allows rapid and reproducible purification of fully active human or guinea pig C3 on a daily basis.

Serum resistance of metacyclic stage Leishmania major promastigotes is due to release of C5b-9.

The mechanism of serum resistance for infective promastigotes of Leishmania major was investigated. Prior results suggested that the mechanism of resistance was mediated at a step after C3 deposition. Equivalent amounts of C3b were deposited on serum-susceptible, noninfective promastigotes harvested from log stage cultures (LOG) and on C-resistant, infective, metacyclic promastigotes (MP) purified from stationary stage cultures. Whereas binding of C9 to LOG was stable during incubation in serum, C9 binding to MP was minimal and unstable, because molecules bound initially to MP were released with continued incubation. Failure to bind C9 was not a result of inability to activate C; the kinetics of C3, C6, and C9 consumption were similar for LOG and MP. Deposition of C5b-7 on MP was stable, indicating that the initial steps in terminal complex formation were intact. Instead, the majority of C5b-9 formed on MP was spontaneously released into the serum as SC5b-9. Residual C5b-9 on MP was released with 1 M NaCl. These data show that developmental modification of the promastigote membrane during transition from a noninfective to an infective stage blocks insertion of lytic C5b-9 into the promastigote membrane.

Generation of C5-dependent bioactivity by tissue-bound anti-BMZ autoantibodies.

We previously reported that complement-binding antibasement membrane zone (BMZ) autoantibodies can mediate complement-dependent directed migration and adherence of leukocytes to the BMZ in cryostat skin sections and that there is heterogeneity in the ability of anti-BMZ autoantibodies to mediate that response. Those observations suggested that directed migration and adherence of leukocytes to the BMZ might be dependent on the amount of complement-activating autoantibody deposited at the BMZ and the extent to which those antibodies could activate complement and generate C5-derived peptides (C5a, C5a des arg). In this study, we have examined the role of autoantibody concentration and C5 in mediating the adherence response. When cryostat skin sections were pretreated with anti-BMZ autoantibodies and subsequently incubated with neutrophils suspended in fresh serum, neutrophils adhered to the BMZ. Adherence was anti-BMZ autoantibody specific and proportional to anti-BMZ autoantibody concentration. To determine the role of C5 in mediating adherence, neutrophils were suspended in increasing concentrations of: 1) fresh serum, 2) heat-inactivated serum, 3) serum pretreated with antihuman C5, 4) serum pretreated with antihuman IgG, 5) C5-depleted serum, 6) purified C5, and 7) C5-depleted serum reconstituted with increasing concentrations of purified C5. The suspensions were then incubated with autoantibody-treated skin sections. The results showed a dose-dependent requirement for fresh serum and for C5-depleted serum reconstituted with increasing doses of C5. Adherence could be detected with C5 concentrations less than 200 ng/ml, which correspond to a C5a/C5a des arg concentration of 10(-8)-10(-9) molar. These results suggest that complement-dependent neutrophil adherence is a highly sensitive method for detecting and quantitating the ability of tissue-deposited anti-BMZ autoantibodies to activate complement and generate C5-derived bioactive peptides, for estimating the amount of C-activating anti-BMZ autoantibody deposited at the BMZ in vivo, and for evaluating the potential role of C-activating anti-BMZ autoantibodies in the pathogenesis of lesions.

A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose.

C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10-12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40-45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.

Multimeric C9 within C5b-9 deposits in unique locations in the cell wall of Salmonella typhimurium.

We have shown previously that multimeric C9 within C5b-9 (C9:C5b-8 greater than 3:1) is needed for killing of a rough strain of Escherichia coli. We now extend these studies using serum sensitive, rough (R) and serum resistant, wild type (WT) strains of Salmonella typhimurium as well as a mutant S. typhimurium strain (TS) with a temperature sensitive mutation in synthesis of keto-deoxy-octulosonate, a constituent within the deep core structure of Salmonella LPS. Both R and TS required multimeric C9 within C5b-9 to be killed. Addition at 37 degrees C of increasing inputs of C9 to TS or R bearing C5b-9 led to a dose-related increase in C9 binding and killing. In contrast, addition of high inputs of C9 to the same strains at 4 degrees C, a procedure that limits the C9:C5b-8 ratio to 1:1, resulted in low C9 binding and minimal killing. Bactericidal C5b-9 formed at 37 degrees C on R and TS with high inputs of C9 co-sedimented with the bacterial outer membrane on sucrose density gradient analysis. Non-bactericidal C5b-9 on R, WT, and TS co-sedimented near the inner membrane, despite the presumed lack of association between these constituents. Whereas 125I C9 within the non-bactericidal pools immunoprecipitate with anti-C5, 125I C9 within bactericidal pools did not immunoprecipitate with anti-C5, anti-C7, or anti-C9. These findings suggest that bactericidal C5b-9 may be deposited in a unique location or configuration within the bacterial cell wall.

Interaction of Neisseria gonorrhoeae with classical complement components, C1-inhibitor, and a monoclonal antibody directed against the Neisserial H.8 antigen.

Strains of Neisseria gonorrhoeae were used to evaluate bactericidal and opsonic properties of McAb 10 directed against the Neisserial outer membrane antigen, H.8. Gonococci were either serum resistant in the absence but serum sensitive in the presence, of McAb 10, or serum sensitive or serum resistant regardless of the presence of McAb 10. Strain JS3, which fell in the former category, was used in subsequent studies. C1 zymogen formed by reassociation of isolated C1 subunits was not directly activated by JS3 in the presence or absence of C1-inhibitor. JS3 thus was unable to directly activate the classical pathway independently of antibody. When purified classical pathway components were used to deposit C3 on JS3 in the absence of serum regulatory proteins or antibodies, added C1-inhibitor reduced C3 binding to background levels. When McAb 10 was present, C3 binding was unaffected by C1-inhibitor. Covalently bound, large molecular weight C3 alpha-chain-gonococcal complexes were disbanded by methylamine release of ester linkages. Released 125I-C3 migrated as C3b without degradation by gonococcal proteases. Purified classical components alone or McAb 10 alone facilitated JS3 killing by neutrophils; when combined, the two provided maximal killing. Levels of McAb 10 that only slightly increase C3 deposition on JS3 are bactericidal in serum and maximally opsonic in combination with purified classical pathway components.

Isolation and characterization of a novel plasma protein which binds to activated C4 of the classical complement pathway.

We report here the isolation and partial characterization of a previously unrecognized protease-sensitive plasma protein identified during the development of a novel protocol for the purification of the second component of human complement (C2). This new protein is physicochemically similar to C2. It coprecipitates with C2 on polyethylene glycol fractionation and specifically binds, like C2, to Sepharose-bound iC4/C4b. Binding occurs both in the presence and absence of C2. The purified protein has a chain structure similar to C2 as determined by sodium dodecyl sulfate-gel electrophoresis in the presence or absence of reducing agent and has a molecular mass of 120 kDa, only somewhat greater than C2 at 95 kDa. Both proteins radioiodinate under similar conditions to the same specific activities with each of two different methods that yield 10-fold disparate results. Quantitative Mancini analysis identifies 300 micrograms/ml of the 120-kDa protein in plasma and serum. The protein is present at normal concentrations in serum from individuals genetically deficient in C2, has no C2 functional activity, and is not cleaved as is C2 when serum complement is activated. Potent monospecific polyclonal anti-serum to each do not cross-immunoprecipitate using standard gel techniques. However, these anti-sera identify epitopes in common by Western blotting. The data presented indicate that the 120-kDa protein is a distinct plasma component and suggest that the protein is not an "immature" form of C2. Initial experiments to delineate a functional role for the 120-kDa protein have demonstrated a consistent inhibition of C1 site generation on EAC4b which is dose-dependent and reversible. Thus, this protein appears to be a new complement regulatory factor.

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization.

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases.

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.

Acquired angioedema: observations on the mechanism of action of autoantibodies directed against C1 esterase inhibitor.

We describe a mechanism of action of autoantibodies reactive with the C1 esterase inhibitor (C1EI) molecule found in three patients with acquired angioedema without associated diseases. All of these patients have a circulating C1EI of lower molecular weight than that of normal control subjects. When native C1EI was added to patient, but not control, plasmas, it was cleaved to a lower molecular weight fragment under conditions that allowed contact system activation. In a partially purified system, patient immunoglobulin preparations impaired stable complex formation between plasmin and C1EI and exaggerated the cleavage of the latter to a lower molecular weight fragment. We propose that the autoantibody does not interfere with the cleavage of the bait sequence of the inhibitor but reduces the availability of the reactive center of the molecule in a way that interferes with the irreversible inhibition of target enzymes. In this way unregulated activation of the kinin and complement pathways occurs, leading to disease manifestations via as yet uncharacterized mediators.

Developmentally regulated expression by Trypanosoma cruzi of molecules that accelerate the decay of complement C3 convertases.

We recently showed that culture-derived metacyclic trypomastigotes (CMT), but not epimastigotes (Epi), of the Miranda 88 strain of Trypanosoma cruzi evade lysis by the human alternative complement pathway because of inefficient binding of factor B to complement component C3b on the parasite surface. These results suggested that CMT and tissue-culture-derived trypomastigotes (TCT), which also activate the alternative pathway poorly, might produce a molecule capable of interfering with factor B binding to C3b. We now demonstrate that CMT and TCT lysates, as well as molecules spontaneously shed from CMT and TCT but not Epi, accelerate decay of 125I-labeled factor Bb from the alternative-pathway C3 convertase (C3bBb) assembled on zymosan or Epi and also accelerate decay of the classical-pathway C3 convertase (C4b2a) on sheep erythrocytes. Parasites metabolically labeled with [35S]methionine spontaneously shed a limited number of radioactive components ranging in molecular mass from 86 to 155 kDa for trypomastigotes and 25 to 80 kDa for Epi. Decay-accelerating activity within supernatants is inactivated by papain and is coeluted with 35S-containing polypeptides on FPLC anion-exchange chromatography, suggesting that the active constituents are protein molecules. Molecules with decay-accelerating activity may explain the developmentally regulated resistance to complement-mediated lysis in infective and vertebrate stages of the T. cruzi life cycle.