Smallpox inhibitor of complement enzymes (SPICE): dissecting functional sites and abrogating activity.

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host's immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity approximately 200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.

Modulation of the formation of the amplification convertase of complement, C3b, Bb, by native and commercial heparin.

Native rat mast cell macromolecular heparin proteoglycan and commercial hog heparin glycosaminoglycan chains inhibit generation of the amplification convertase, C3b, Bb. The inhibitory action of heparin is not due to chelation of magnesium. Heparin is most active in inhibiting convertase formation on cellular intermediates formed with the lowest C3b input and developed with the highest B concentration, thereby suggesting the receptor site for B on C3b as the point of heparin action. This interpretation is consistent with the demonstration that heparin prevents B utilization during the fluid phase interaction of C3b, B, and D. Inhibition is observed also when C3b,Bb generation takes place on cellular intermediates in the presence of P or C3NeF, which yield stabilized forms of the convertase. 50 times the concentration of heparin required to inhibit convertase generation does not accelerate the decay of the unstabilized or the C3NeF-stabilized convertases and has only a modest effect on the P-stabilized convertase. An additional effect of heparin is to impair beta1H-mediated decay-dissociation of C3b,Bb. The concentration of native or commercial heparin which prevents convertase formation is in the same range as that required for the demonstration of its anti-coagulant and anti-thrombin III cofactor activities. The additional finding that this inhibitory action of heparin can be expressed by the isolated mast cell granule suggests that native heparin may contribute to the modulation of the amplification pathway of complement.

Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes.

Decay-accelerating factor (DAF), extracted from the stroma of human erythrocytes, was purified to homogeneity and incorporated into the membrane of sheep red cell complement intermediates, where its functional properties were analyzed. Incorporation of DAF into the cell membranes was temperature dependent, took place on pronase- or trypsin-treated erythrocytes, and did not depend on prior deposition of antibody, C1 or C4. Serum lipoproteins (high and low density) effectively inhibited DAF incorporation, but had no effect on the activity of DAF after its association with the cell membrane. The incorporated DAF could not be removed from the red cell surface by repeated washings in the presence of high salt concentration but was solubilized when the stroma were extracted with 0.1% Nonidet P-40. The presence of DAF in the membrane of EA did not affect the deposition of C1 and C4, but as few as 10(2) DAF molecules per cell profoundly inhibited the assembly of C3 and C5 convertases of both the classical and alternative pathways. The DAF inhibitory effect on EAC14 or EAC43 was not overcome by supplying an excess of C2 or factor B, but the alternative pathway C3 convertase could be assembled in the presence of Ni++, or nonphysiological concentrations of Mg++, which enhances the binding affinity of factor B for C3b. The DAF effect on EAC14 or EAC143 was entirely reversed by treating the cells with specific anti-DAF antibodies, showing that DAF did not alter the structure of C4b or C3b. Taken together, the experimental evidence suggests that DAF interacts directly with membrane-bound C3b or C4b and prevents subsequent uptake of C2 and factor B. DAF can function only within the cell membrane. Indeed, the decay dissociation of the C4b2a enzyme on DAF-containing sheep intermediates was not changed by varying the cell concentration. DAF-treated EA had no influence on the decay of nontreated EAC142 present in the same mixture. Moreover, the inhibitory activity of intact human erythrocytes on C4b2a was not blocked by antibodies to DAF, but was abolished by antibodies to the C3b/C4b receptor (CR1). When incorporated into the membrane of rabbit erythrocytes, human DAF inhibited their lysis by human complement. In conclusion, on the basis of these and previous results, it appears that DAF plays a central role in preventing the amplification of the complement cascade on host cell surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanism of action of decay-accelerating factor (DAF). DAF inhibits the assembly of C3 convertases by dissociating C2a and Bb.

DAF is a 70,000-Mr membrane protein that inhibits the amplification of the complement cascade on the cell surface, and protects cells from damage by complement. The precise mechanism of action of DAF is not entirely clear. Purified DAF was incorporated into the membrane of EAC4b cells. EAC4b2 and EDAF AC4b2 cells were prepared with radiolabeled C2. The same amount of labeled C2 bound to both cells, showing that DAF does not prevent the binding of C2 zymogen to C4b. After adding Cl, the radioactivity of bound C2 dissociated more rapidly from EDAF AC4b cells than from EAC4b cells. In EAC4b cells, bound C2 was converted to C2a, which gradually dissociated into the supernatants. In the DAF-treated cells, on the other hand, a large amount of C2a rapidly appeared in the supernatants and only a small amount of C2a remained on the cells. In a similar experiment using EhuAC4b, DAF on human erythrocyte membrane also dissociated the C2a from the cells. These results were confirmed by hemolytic assay and the accelerated decay of C2a caused the rapid depletion of C2 from the fluid phase. In addition, we found that DAF functions on the alternative pathway C3 convertase, C3bBb in the same manner. Thus, DAF, which associates with C4b and C3b in the membrane, acts on C2a and Bb, but not on intact C2 and B, and dissociates them rapidly from the binding sites, thereby preventing the assembly of the classical and alternative pathways C3 convertases.

Smallpox inhibitor of complement enzymes (SPICE): regulation of complement activation on cells and mechanism of its cellular attachment.

Despite eradication of smallpox three decades ago, public health concerns remain due to its potential use as a bioterrorist weapon. Smallpox and other orthopoxviruses express virulence factors that inhibit the host's complement system. In this study, our goals were to characterize the ability of the smallpox inhibitor of complement enzymes, SPICE, to regulate human complement on the cell surface. We demonstrate that SPICE binds to a variety of cell types and that the heparan sulfate and chondroitin sulfate glycosaminoglycans serve as attachment sites. A transmembrane-engineered version as well as soluble recombinant SPICE inhibited complement activation at the C3 convertase step with equal or greater efficiency than that of the related host regulators. Moreover, SPICE attached to glycosaminoglycans was more efficient than transmembrane SPICE. We also demonstrate that this virulence activity of SPICE on cells could be blocked by a mAb to SPICE. These results provide insights related to the complement inhibitory activities of poxviral inhibitors of complement and describe a mAb with therapeutic potential.

A circulating inhibitor of fluid-phase amplification. C3 convertase formation in systemic lupus erythematosus.

C3 nephritic factor (C3NeF) was used to assess the formation of the fluid-phase amplification convertase, C3b,Bb, in 37 serum specimens from 24 patients with systemic lupus erythematosus (SLE). C3b,Bb formation was measured by the concentration of Ba, released when C3b,B is activated. Incubation of normal human serum (NHS) with C3NeF accelerates C3b amplification loop turnover with the formation of large quantities of C3b,Bb. In contrast, sera from 22 of 24 patients with SLE formed little or no convertase when incubated with C3NeF. C3 conversion to C3b was commensurately reduced. The inhibition could not be attributed to depressed serum concentrations of C3, factor B, or classical pathway components. Inhibitor present in excess could be demonstrated in 23 of 34 specimens of SLE serum by mixing experiments. The spontaneous convertase formation that occurs when a portion of the serum H is inactivated with F(ab')2 anti-H was also shown to be inhibited in SLE serum. The inhibition was found, however, to be H dependent in that convertase formation was normal in SLE serum depleted of H. It is concluded that the C3b in most SLE sera is unusually susceptible to inactivation by H, but a functional abnormality was not demonstrable in either C3 or H isolated from SLE serum. The inhibition could be simulated in NHS by addition of heparin, 100 micrograms/ml. In vivo, inhibition of convertase formation could interfere with the solubilization and disposal of immune complexes by reducing the deposition of C3b on the immune complex lattice.

Evidence for arginine residues in the immunoglobulin-binding sites of human Clq.

The immune complex binding activity of human Clq was lost following treatment of the protein with the arginine-selective reagents cyclohexane 1,2-dione and phenylglyoxal. Both inactivations followed pseudo-first-order kinetics. The affinity of Clq for immune complexes was reduced 7-fold following cyclohexane-1,2-dione treatment, and could be substantially restored by treatment of the modified protein with hydroxylamine. Heat-aggregated IgG protected Clq against inactivation by both reagents. Incorporation of 25 molecules of [7-14C]phenylglyoxal per Clq molecule completely inactivated the protein. These data are consistent with the presence of arginyl residues in the immunoglobulin recognition sites of human Clq.

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.

New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin.

p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.

Heparin prevents formation of the human C3 amplification convertase by inhibiting the binding site for B on C3b.

Fluid-phase heparin prevents generation of the C3 amplification convertase of human complement, C3b, Bb most likely by inhibiting the formation of the bimolecular complex between cell-bound C3b and B. The effect of heparin on the binding of B to C3b was examined using 125I-labelled B and C3b-bearing sheep erythrocytes (EsC3b). In the absence of heparin, B bound to EsC3b with an affinity of 0.5-1 X 10(6) M-1 in the presence of 5 mM Mg2+. Incremental amounts of heparin (100-700 micrograms/10(7) EsC3b) inhibited the binding of 125I-B to C3b in a dose-dependent manner. Scatchard analysis of the binding data in the presence of four inhibitory concns of heparin revealed that heparin did not affect the binding affinity of B for C3b but decreased the number of C3b sites recognized by B on the cells. No inhibition of binding occurred in the presence of totally (N- and O-) desulfated heparin which has no anticomplementary activity. These results demonstrate that heparin prevents generation of the C3 amplification convertase by binding to cell-bound C3b and masking the binding site for B on C3b.

The effect of ethylenedinitrilotetraacetic acid (EDTA) on the reaction between the guinea-pig C5 convertase and guinea-pig C5.

Guinea-pig C5 was reacted with EAC1423 in the washed-cell intermediate assay in the presence of glucose gelatin veronal buffer (GGVB), Zn2+-GGVB (0.025 mM), GGVB2+ containing Ca2+ and/or Mg2+ or EDTA (0.013 M)-GGVB. The EDTA inhibited the formation of competent SAC14235, while Ca2+ and/or Mg2+ had a slight enhancing effect compared to GGVB alone and Zn2+ gave a four-fold increase. Similar results were obtained by using human C5 with guinea-pig C5 convertase and functionally pure guinea-pig C6, C7, C8 and C9. When guinea-pig C6 was incorporated into these various reaction mixtures with guinea-pig C5, its addition markedly reduced the inhibition by EDTA, while Zn2+ still showed an enhancing effect. These results demonstrate that EDTA inhibited formation of competent SAC14235 by preventing activation of C5. The association of C6 with C5 can partially overcome the inhibition of C5 conversion by EDTA and may account for C5 activity in reaction mixtures containing C-EDTA.

Detection of intermediary Clr with complete active site, using a synthetic proteinase inhibitor.

The synthetic proteinase inhibitor, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), strongly suppressed activation of Clr at 37 degrees C, causing 50% inhibition at 0.03 mM. To clarify whether the inhibitor was incorporated into the active site of intermediary Clr formed during the incubation, determination of the active site was tried using this inhibitor. Consequently, release of amidinonaphthol equimolar with the amount of Clr used was observed in the early period of incubation, in which the activation to Clr- was about 5%. These results indicate that intermediary Clr already has a complete active site.

Prekallikrein activation, C1 esterase inhibitor, and factor XII as predictors of adverse reaction to contrast media. A prospective study.

The susceptibility of 152 patients to idiosyncratic reactions resulting from the administration of radiographic contrast media was studied. The rate of activation of plasma prekallikrein was measured in samples taken from these patients before they received contrast agents. Kallikrein inhibitor and factor XII levels were also determined. The tests were of no value in selecting the ten patients who subsequently experienced mild reactions. However, the possibility remains that one or more of the tests may have predictive value for patients who experience moderate or severe reactions.

Circulating proalbumin associated with a second case of antitrypsin Pittsburgh.

We report here the conclusive identification of circulating proalbumin of normal N-terminal sequence (Arg Gly Val Phe Arg Arg Asp Ala) in a second child with the alpha 1-antitrypsin Pittsburgh 358 Met-->Arg mutation. As in the first case, the proalbumin made up 3-5% of the total serum albumin. The finding of proalbumin associated with a second de novo mutation at the inhibitory site bait of antitrypsin confirms our earlier hypothesis; that antitrypsin Pittsburgh was acting as a specific intracellular inhibitor of the hepatic proalbumin convertase and that antitrypsin Pittsburgh could be used as a probe to identify the proprotein convertase.

[Changes in the ultrastructure of subcomponent C1q of human complement during spontaneous inactivation in diluted solutions]. / Izmeneniia ul'trastruktury subkomponenta C1q komplememnta cheloveka pri spontannoi inaktivatsii v razbavlennykh rastvorakh.

Dilution of human serum or solutions of highly purified subcomponent C1q of human complement results in the drop of C1q activity. Electron microscopy of highly purified subcomponent C1q revealed that a certain part of molecules has a changed ultrastructure and C1q subunits are dissociated. As the preparations for electron microscopy have been obtained from dilute solutions, the changes in the ultrastructure and C1q inactivation should be interrelated phenomena. The conformational liability of the C1q structure is supposed to have a functional role.

[Binding of activated C3b component with complement effector]. / Sviazyvanie aktivirovannogo komponenta C3b s éffektorami komplementa.

Various nucleophilic agents (acceptors) react with thiolester group of nascent activated fragment (C3b) of the third complement component. The C3b-acceptors binding prevents transformation of C3 convertase to C5 convertase and results in inhibition of the cell-target lysis. A convenient method of monitoring the EAC142 to EAC1423 transformation was elaborated. Character of the inhibition suggests that the covalent binding follows a stage of the reversible C3b-acceptor complex formation. The method allows to determine the maximum of inhibition of the C5 convertase formation and the dissociation constant of the reversible C3b-acceptor complex, which reflects the C3b affinity to this acceptor.

[Effect on human complement of blastolysin and the glycopeptide (MDP and GMDP) and carbohydrate fragments of peptidoglycans]. / Deistvie na komplement cheloveka blastolizina, a takzhe glikopeptidnykh (MDP, GMDP) i uglevodnykh fragmentov peptidoglikanov.

Along with complement activation by the classical pathway, blastolysin, an antitumor and adjuvant preparation of Lactobacillus bulgaricus peptidoglycans, effectively inhibits the transformation of C3 in to C5 convertase. Values of inhibition maximum and dissociation constants of the reversible C3b-acceptor complex for blastolysin and main immunological active structural moieties of peptidoglycans (GMDP, MDP) and their inactive carbohydrate components (N-acetylglucosaminyl-N-acetylmuramic acid, N-acetylglucosamine, and N-acetylmuramic acid) have been determined. Immunostimulator concentrations for blastolysin, GMDP, and MDP in inhibition of the C5 convertase formation (C3b binding) correlate with their doses in vivo (animal blood), displaying antitumor activity.

rhC1INH: a new drug for the treatment of attacks in hereditary angioedema caused by C1-inhibitor deficiency.

Recombinant human C1 esterase inhibitor (rhC1INH) (Ruconest(®), Pharming) is a new drug developed for the relief of symptoms occurring in patients with angioedema due to C1-inhibitor deficiency. Pertinent results have already been published elsewhere; this article summarizes the progress made since then. Similar to the purified C1-inhibitor derived from human plasma, the therapeutic efficacy of rhC1INH results from its ability to block the actions of enzymes belonging to the overactivated bradykinin-forming pathway, at multiple locations. During clinical trials into the management of acute edema, a total of 190 subjects received recombinant C1-inhibitor by intravenous infusion on 714 occasions altogether. Dose-ranging efficacy studies established 50 U/kg as the recommended dose, and demonstrated the effectiveness of this agent in all localizations of hereditary angioedema attacks. Studies into the safety of rhC1INH based on 300 administrations to healthy subjects or hereditary angioedema patients followed-up for 90 days have not detected the formation of autoantibodies against rhC1INH or IgE antibodies directed against rabbit proteins, even after repeated administration on multiple occasions. These findings met favorable appraisal by the EMA, which granted European marketing authorization for rhC1INH. Pharming is expected to file a biological licence with the US FDA by the end of 2010 to obtain marketing approval in the USA. The launch of rhC1INH onto the pharmaceutical market may represent an important progress in the management of hereditary angioedema patients.