Affinity adsorption of phospholipase A1 with designed ligand binding to catalytic pocket.

An affinity ligand was designed from 1-aminocyclohexane based on the crystal structure of Streptomyces albidoflavus phospholipase A1 (saPLA1) by using Discovery Studio software. The molecular docking results indicated that the designed ligand could interact with the active pocket of saPLA1. Epichlorohydrin, cyanuric chloride and 1-aminocyclohexane were used to synthesize the affinity ligand, which was composed to Sepharose beads. The density of the ligand on Sepharose beads was 22.5 ± 1.1 µmol/g wet gel. Adsorption analysis of the sorbent indicated the maximum adsorption (Qmax) of the enzyme was 10.7 ± 0.29 mg/g and the desorption constant (Kd) was 426.6 ± 29.7 µg/mL. The sorbent could bind the enzyme in the supernatant of disrupted recombinant Escherichia coli through one step of affinity adsorption. After the optimization of the purification process, a single band was obtained at approximately 30 kDa, which was confirmed as saPLA1 by the matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry and activity assay. The purity of the isolated enzyme was about 96.6% with the purify fold at 7.62, and the activity recovery was 52.5%.

Cellular localization, cloning and expression of Leishmania braziliensis Phospholipase A1.

Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A1 (PLA1) activity, an enzyme that catalyses extensive deacylation of phospholipids like phosphatidylcholine. In order to deepen the knowledge about L. braziliensis PLA1, the cloning and expression of the gene that codifies for this enzyme was carried out in a baculovirus expression system with the obtaintion of a purified recombinant protein that displayed PLA1 activity. Given that this is the first molecular and functional protein characterization of a PLA1 in the Leishmania genus, we also performed a phylogenetic analysis of this gene throughout 12 species whose genome sequences were available. The results presented here will contribute to increase the knowledge about trypanosome phospholipases, which could be novel and valuable as potential targets to fight neglected diseases like Leishmaniasis.

Purification and biochemical characterization of VesT1s, a novel phospholipase A1 isoform isolated from the venom of the greater banded wasp Vespa tropica.

Vespa tropica, a social wasp locally found in Thailand is responsible for many out off the record accidental stings due to close encounters with human activities and because of the animal's highly potent venom. Phospholipase (PLA) is one of the major proteins commonly found in insect venom. In this work, V. tropica phospholipase was successfully isolated, purified and characterized. Three isoforms PLAs have been purified using reversed phase HPLC, and are named VesT1s (VesT1.01a, VesT1.01b and VesT1.02). They are not glycoproteins. VesT1.01s has a molecular weight of 33.72 kDa while for VesT1.02 a mass of 34 kDa was found. The deduced sequence of the mature VesT1.02 protein is composed of 301 amino acid residues (1005 bp), including the catalytic triad (Ser-His-Asp), which is similar to other wasp venom PLAs. The 12 cysteine residues found are conserved among venom PLA1. They form six disulfide bonds, and therefore have no free sulfhydryl groups. Based on homology modelling, VesT1.02 belongs to the α/ß hydrolase fold family. Its structure is composed of 10 ß-sheets and 11 α-helixes, characterized by a ß-strand/εSer/α-helix structural motif, which contains the Gly-X-Ser-X-Gly consensus sequence. The shortened lid and shortened ß9 loop, which play important roles in substrate selectivity, cause this enzyme to only exhibit PLA activity. Moreover, these PLAs have been shown to be highly thermally stable after heating at 100 °C for 5 min. We propose that an inserted Pro residue might be involved in this high thermo-stability.

Molecular cloning, expression and IgE-immunoreactivity of phospholipase A1, a major allergen from Polybia paulista (Hymenoptera: Vespidae) venom.

Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P. paulista venom. The cDNA of Poly p 1 was extracted from venom glands and then cloned, and further expression of the recombinant allergen (rPoly p 1) was achieved in Escherichia coli BL21 (DE3) cells. Purification of rPoly p 1 was performed using immobilized Ni2+ metal affinity chromatography. Also, a single-step chromatographic method allowed the purification of native Poly p 1 (nPoly p 1) from the wasp's venom glands. We used western blotting to evaluate IgE-reactivity of the sera from 10 P. paulista venom-allergic patients to rPoly p 1 and nPoly p 1. High levels of insoluble rPoly p 1 were obtained during heterologous expression. After solubilization of inclusion bodies and purification of the recombinant protein, a unique band of ∼34 kDa was detected in SDS-PAGE analysis. Allergen-specific IgE (sIgE) from allergic patients' sera recognized rPoly p 1, nPoly p 1 and crude venom extract to a similar extent. Our results showed that rPoly p 1 could be used for development of component-resolved diagnosis (CRD) and molecular-defined SIT of P. paulista venom allergy.

Recombinant Phospholipase A1 of the Outer Membrane of Psychrotrophic Yersinia pseudotuberculosis: Expression, Purification, and Characterization.

The pldA gene encoding membrane-bound phospholipase A1 of Yersinia pseudotuberculosis was cloned and expressed in Escherichia coli cells. Recombinant phospholipase A1 (rPldA) was isolated from inclusion bodies dissolved in 8 M urea by two-stage chromatography (ion-exchange and gel-filtration chromatography) as an inactive monomer. The molecular mass of the rPldA determined by MALDI-TOF MS was 31.7 ± 0.4 kDa. The highly purified rPldA was refolded by 10-fold dilution with buffer containing 10 mM Triton X-100 and subsequent incubation at room temperature for 16 h. The refolded rPldA hydrolyzed 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in the presence of calcium ions. The enzyme exhibited maximal activity at 37°C and nearly 40% of maximal activity at 15°C. The phospholipase A1 was active over a wide range of pH from 4 to 11, exhibiting maximal activity at pH 10. Spatial structure models of the monomer and the dimer of Y. pseudotuberculosis phospholipase A1 were constructed, and functionally important amino acid residues of the enzyme were determined. Structural differences between phospholipases A1 from Y. pseudotuberculosis and E. coli, which can affect the functional activity of the enzyme, were revealed.

Hydrolysis of plasmalogen by phospholipase A1 from Streptomyces albidoflavus for early detection of dementia and arteriosclerosis.

OBJECTIVES: To obtain an ethanolamine plasmalogen (PlsEtn)-hydrolyzing enzyme and to develop an assay that would help determine PlsEtn concentrations in human serum as an indicator of Alzheimer-type dementia and of arteriosclerosis. RESULTS: Phospholipase A1s, SaPLA1 and SvPLA1 from, respectively, Streptomyces albidoflavus NA297 and S. avermitilis JCM5070-but not phospholipase B from Streptomyces sp. NA684, PLA2-Nagase from S. avermitilis, PLA2IIL from S. violaceoruber nor LIPOMOD 699L (porcine phospholipase)-hydrolyzed choline plasmalogen (PlsCho) and PlsEtn (PlsCho preferred over PlsEtn). Using a combination of SaPLA1, lysoplasmalogen-specific phospholipase D (LyPls-PLD), with amine oxidase, an end-point assay was developed for measuring serum PlsEtn concentration. The standard curve, generated using various amounts of PlsEtn in this assay, was linear between 0 and 0.2 mM. PlsEtn concentrations in forty-seven serum samples, determined independently by this enzyme-based assay and (125)I-HPLC method, exhibited a linear relationship, indicating that the assay is suitable for fast and accurate measurement of serum PlsEtn concentration. CONCLUSIONS: An assay, developed using SaPLA1, LyPls-PLD, and AOX, selectively measured PlsEtn levels in blood samples. This assay could be a useful diagnostic tool for early stage detection of diseases such as Alzheimer-type dementia and arteriosclerosis.

Purification and structural characterisation of phospholipase A1 (Vespapase, Ves a 1) from Thai banded tiger wasp (Vespa affinis) venom.

The Thai banded tiger wasp (Vespa affinis) is one of the most dangerous vespid species in Southeast Asia, and stinging accidents involving this species still cause fatalities. In the present study, four forms of V. affinis phospholipase A(1) were identified through a proteomics approach. Two of these enzymes were purified by reverse-phase chromatography, and their biochemical properties were characterised. These enzymes, designated Ves a 1s, are not glycoproteins and exist as 33441.5 and 33474.4 Da proteins, which corresponded with the 34-kDa band observed via SDS-PAGE. The thermal stabilities of these enzymes were stronger than snake venom. Using an in vivo assay, no difference was found in the toxicities of the different isoforms. Furthermore, the toxicity of these enzymes does not appear to be correlated with their PLA(1) activity. The cDNAs of the full-length version of Ves a 1s revealed that the Ves a 1 gene consists of a 1005-bp ORF, which encodes 334 amino acid residues, and 67- and 227-bp 5' and 3' UTRs, respectively. The two isoforms are different by three nucleotide substitutions, resulting in the replacement of two amino acids. Through sequence alignment, these enzymes were classified as members of the pancreatic lipase family. The structural modelling of Ves a 1 used the rat pancreatic lipase-related protein 2 (1bu8A) as a template because it has PLA(1) activity, which demonstrated that this enzyme belongs to the α/ß hydrolase fold family. The Ves a 1 structure, which is composed of seven α-helixes and eleven ß-strands, contains the ß-strand/ɛSer/α-helix structural motif, which contains the Gly-X-Ser-X-Gly consensus sequence. The typical surface structures that play important roles in substrate selectivity (the lid domain and the ß9 loop) were shortened in the Ves a 1 structure, which suggests that this enzyme may only exhibit phospholipase activity. Moreover, the observed insertion of proline into the lid domain of the Ves a 1 structure is rare. We therefore propose that this proline residue might be involved in the stability and activity of Ves a 1s.

Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST). Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.

The Rickettsia prowazekii ExoU homologue possesses phospholipase A1 (PLA1), PLA2, and lyso-PLA2 activities and can function in the absence of any eukaryotic cofactors in vitro.

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA(2) and lyso-PLA(2) activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA(1) activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.

Crystallization and preliminary X-ray diffraction analysis of phospholipase A1 isolated from hornet (Vespa basalis) venom.

Phospholipase A(1) (PLA(1)) isolated from the black-bellied hornet (Vespa basalis) catalyzes the hydrolysis of emulsified phospholipids in addition to the potent haemolytic activity responsible for its lethal effect. In this study, the crystallization and preliminary crystallographic analysis of PLA(1) from hornet venom with a molecular weight of 32 kDa are reported. PLA(1) was crystallized at 277 K using PEG 4000 as precipitant and a 96.5% complete native data set was collected from a frozen crystal to 2.5 A resolution at 100 K with an overall R(merge) of 6.8%. The crystal belongs to the triclinic space group P1, with unit-cell parameters a = 57.2, b = 70.2, c = 81.6 A, alpha = 107.0, beta = 109.9, gamma = 100.9 degrees . In each asymmetric unit, three or four subunits of PLA(1) are present according to the calculation of the solvent content.

Determination of membrane protein molecular weights and association equilibrium constants using sedimentation equilibrium and sedimentation velocity.

Regulated molecular interactions are essential for cellular function and viability, and both homo- and hetero-interactions between all types of biomolecules play important cellular roles. This chapter focuses on interactions between membrane proteins. Knowing both the stoichiometries and stabilities of these interactions in hydrophobic environments is a prerequisite for understanding how this class of proteins regulates cellular activities in membranes. Using examples from the authors' work, this chapter highlights the application of analytical ultracentrifugation methods in the determination of these parameters for integral membrane proteins. Both theoretical and practical aspects of carrying out these experiments are discussed.