Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.

The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases.

Interfacial recognition by bee venom phospholipase A2: insights into nonelectrostatic molecular determinants by charge reversal mutagenesis.

The basis for tight binding of bee venom phospholipase A2 (bvPLA2) to anionic versus zwitterionic phospholipid interfaces is explored by charge reversal mutagenesis of basic residues (lysines/arginines to glutamates) on the putative membrane binding surface. Single-site mutants and, surprisingly, multisite mutants (2-5 of the 6 basic residues mutated) are fully functional on anionic vesicles. Mutants bind tightly to anionic vesicles, and active-site substrate and Ca2+ binding are not impaired. Multisite mutants undergo intervesicle exchange slightly faster than wild type, especially in the presence of salt. It is estimated that electrostatic contribution to interfacial binding is modest, perhaps 2-3 kcal/mol of the estimated 15 kcal/mol. Elution properties of bvPLA2 from HPLC columns containing solid phases of tightly packed monolayers of phosphocholine amphiphiles suggest that ionic effects provide a modest portion of the interfacial binding energy and that this contribution decreases as the number of cationic residues mutated is increased. These results are consistent with the observation that Gila monster venom PLA2 (Pa2), which is homologous to bvPLA2, has high activity on anionic vesicles despite the fact that it has only a single basic residue on its putative interfacial recognition face. Results with bvPLA2 mutants show that manoalogue and 12-epi-scalaradial inactivate bvPLA2 by modification of K94. Also, deletion of the large beta-loop (residues 99-118) is without consequence for interfacial binding and catalysis of bvPLA2. All together, the preferential binding of bvPLA2 to anionic vesicles versus phosphatidylcholine vesicles is mainly due to factors other than electrostatics. Therefore hydrogen-bonding and hydrophobic interactions must provide a major portion of the interfacial binding energy, and this is consistent with recent spectroscopic studies.

Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis.

Although the available evidence suggests that whereas the caspase family plays a major role in apoptosis, they are not the sole stimulators of death. A random yeast two-hybrid screen of a lymphocyte cDNA library (using caspase-3 as the bait) found an interaction between caspase-3 and the regulatory subunit Aalpha of protein phosphatase 2A. This protein was found to be a substrate for caspase-3, but not caspase-1, and could compete effectively against either a protein or synthetic peptide substrate. In Jurkat cells induced to undergo apoptosis with anti-Fas antibody, protein phosphatase 2A (PP2A) activity increased 4.5-fold after 6 h. By 12 h, the regulatory Aalpha subunit could no longer be detected in cell lysates. There was no change in the amount of the catalytic subunit. The effects on PP2A could be prevented by the caspase family inhibitors acetyl-Asp-Glu-Val-Asp (DEVD) aldehyde or Ac-DEVD fluoromethyl ketone. The mitogen-activated protein (MAP) kinase pathway is regulated by PP2A. At 12 h after the addition of anti-Fas antibody, a decrease in the amount of the phosphorylated forms of MAP kinase was observed. Again, this loss of activated MAP kinase could be prevented by the addition of DEVD-cho or DEVD-fmk. These data are consistent with a pathway whereby induction of apoptosis activates caspase-3. This enzyme then cleaves the regulatory Aalpha subunit of PP2A, increasing its activity. These data show that the activated PP2A will then effect a change in the phosphorylation state of the cell. These data provide a link between the caspases and signal transduction pathways.

Use of an imperfect neutral diluent and outer vesicle layer scooting mode hydrolysis to analyze the interfacial kinetics, inhibition, and substrate preferences of bee venom phospholipase A2.

Interfacial catalytic constants for bee venom phospholipase A2 (bvPLA2) have been obtained for its action on vesicles of the anionic phospholipid 1,2-dimyristoylphosphatidylmethanol (DMPM) in the highly processive scooting mode. Spectroscopic measurements which directly measure transbilayer movement of membrane components show that this exchange does not occur in anionic vesicles that have undergone complete bvPLA2-catalyzed hydrolysis of all phospholipids in the outer vesicle monolayer. 3-Hexadecyl-sn-glycero-1-phosphocholine (D-LPC) is an adequate neutral diluent for bvPLA2, which is defined as an amphiphile that forms an aggregate to which enzyme binds but neutral diluent molecules bind weakly in the enzyme's active site. D-LPC has weak affinity for the active site of bvPLA2, and theory and protocols are developed that allow its use to determine equilibrium dissociation constants for competing active site ligands. Some of the properties of bvPLA2 are shared by other 14 kDa PLA2s. (1) Ca2+ is required for binding of ligands to the active site but not for the binding of enzyme to the interface. (2) bvPLA2 does not significantly discriminate between phospholipids with different polar head groups or acyl chains. (3) bvPLA2 does not bind to phosphatidylcholine vesicles, and binding occurs if anionic amphiphiles are present in the vesicle. Novel features of bvPLA2 include the following: (1) Neutral diluents for other 14 kDa phospholipases A2 are not neutral diluents for bvPLA2. (2) Saturation of the active site with a variety of different ligands does not completely prevent histidine alkylation by 2-bromo-4'-nitroacetophenone, and Ca2+ binding does not change the rate of histidine alkylation. Finally, the carbohydrate portion of bvPLA2 does not alter the interfacial catalytic properties of the enzyme. Kinetic analysis of bvPLA2 in the scooting mode together with previous studies with other 14 kDa PLA2s provides a paradigm for the quantitative analysis of interfacial catalysis.

Comparison of the antibody response to bee venom phospholipase A2 induced by natural exposure in humans or by immunization in mice.

Two human and twelve murine monoclonal antibodies directed against the main bee venom allergen phospholipase A2 (PLA) were evaluated for their fine specificity of binding to antigen and their ability to inhibit the enzymatic activity of the antigen. Antibodies were induced by natural exposure of beekeepers to bee venom or immunization of mice via different methods. Both human monoclonal antibodies (hmAbs) were previously shown to recognize the native three-dimensional conformation of PLA and are directed against discontinuous epitopes which include lysine residue at position 25 as a contact residue. In contrast, six of the murine monoclonal antibodies (mmAbs) bind to the denatured structure of the protein as determined by enzyme-linked immunosorbent assay. The epitopes recognized are located near the C-terminal end (n = 8), in the centre of the polypeptide (n = 1), near the N-terminal end (n = 1) or include the carbohydrate part (n = 2) of the PLA molecule. The capacity of the antibodies to modify the enzymatic activity was also determined. The hmAbs significantly inhibit the enzyme (70-79%), whereas the mmAbs produced various degrees of inhibition (39-100%). Since the X-ray structure of PLA is known, the epitopes can be visualized in the context of the three-dimensional structure of the antigen. A qualitative correlation was found between the location of epitopes and the inhibition pattern. Strong inhibition was seen with those antibodies that recognize epitopes that lie on the surface of the enzyme that is thought to contact the phospholipid bilayer. The results show that even though both hmAbs and most mmAbs inhibit the enzymatic activity of PLA, the antigen-binding properties of antibodies from different species raised after different routes of immunization differ significantly. Thus, detailed epitope mapping studies using murine antibodies prepared by artificial immunization may have limited value in predicting epitope patterns relevant to an antibody response to allergens in humans naturally exposed to antigen/allergen.

Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87.

In bee venom phospholipase A2, histidine-34 probably functions as a Brønsted base to deprotonate the attacking water. Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34. We have prepared mutants at these positions and studied their kinetic properties. The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis. The pKa of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone. The pKa is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pKa is unchanged. These pKas are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong. The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.

A link between catalytic activity, IgE-independent mast cell activation, and allergenicity of bee venom phospholipase A2.

The molecular and cellular mechanisms controlling Ab isotype selection following encounter of a given Ag are still unclear, although the regulatory role of cytokines is established. In the present study we explored the possibility that the nonimmunologic interaction of an allergen with cells of the innate immune system might result in a release of mediators that promote IgE isotype selection in adaptive responses. Using the bee venom allergen phospholipase A2 (PLA2) and a mutant variant lacking enzymatic function, we show that PLA2, but not its catalytically inactive variant, is able to induce IgE-independent mediator release, including IL-4, from rodent mast cells. Assessing the in vivo relevance of these observations, we find that repeated injections of low doses of active enzyme into mice induce the synthesis of high levels of PLA2-specific IgE, while immunization with the inactive form yields no detectable IgE response. Both Ags were similarly immunogenic when high doses of Ag were used for immunization. These findings suggest that mast cells might be a source of IL-4 at the onset of specific immunity against sources of allergens such as bee venom that contain PLA2 and support the concept that the biologic action of an Ag on cells of the innate immune system can play a role in determining adaptive immune responses.

Antigenic surface of the bee venom allergen phospholipase A2. Structural functional analysis of human IgG4 antibodies reveals potential role in protection.

In the human immune response to the main bee venom allergen phospholipase A2 (PLA2), Abs of the IgG4 isotype are associated with protection. The antigenic sites of PLA2 that are recognized by two mAb of this isotype have been analyzed at the molecular level. The interaction of PLA2 with these two Abs has previously been shown to depend on the conformation of the Ag and to be sensitive to lysine modifications. Therefore, genetically engineered Ag displaying single point mutations of lysine residues were generated. Nine out of twelve surface-exposed lysine residues were substituted by glutamate or glutamine, and the effect of individual mutations on mAb binding was analyzed in the context of the folded Ag. Substitution of lysine at position 25 with either glutamate or glutamine completely abrogates binding to both mAb. All other mutants do not show a difference in binding when compared with wild-type Ag. Probing of sera from bee keepers with lysine 25 mutants reveals that a significant proportion of serum Abs of the IgG4 isotype display specificity for this epitope, suggesting that this represents a major B cell-antigenic site in hyperimmune individuals. We further found that both mAbs inhibit the catalytic activity of PLA2, a property that may account for the protective role of IgG4 Abs in hyperimmune individuals.

High-level expression in Escherichia coli and rapid purification of enzymatically active honey bee venom phospholipase A2.

Bee venom phospholipase A2 (BV-PLA2) is a hydrolytic enzyme that specifically cleaves the sn-2 acyl bond of phospholipids at the lipid/water interface. The same enzyme is also believed to be responsible for some systemic anaphylactic reactions in bee venom sensitized individuals. To study the structure/function relationships of this enzyme and to define the molecular determinants responsible for its allergenic potential, a synthetic gene encoding the mature form of BV-PLA2 was expressed in Escherichia coli. This enzyme was produced as a fusion protein with a 6xHis-tag on its amino-terminus yielding 40-50 mg of fusion protein per 1 of culture after metal ion affinity chromatography. A kallikrein protease recognition site was engineered between the 6xHis-tag and the amino-terminus of the enzyme allowing isolation of the protein with its correct N-terminus. Recombinant affinity purified BV-PLA2 was refolded, purified to homogeneity, and cleaved with kallikrein, resulting in a final yield of 8-9 mg of active enzyme per 1 of culture. The enzymatic and immunological properties of the recombinant BV-PLA2 are identical to enzyme isolated from bee venom indicating a native-like folding of the protein.