Insect venom phospholipases A1 and A2: Roles in the envenoming process and allergy.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus Lipase, a Comparative Structural Study.

Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 µM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.

Improving the catalytic properties of immobilized Lecitase via physical coating with ionic polymers.

Lecitase Ultra has been immobilized on cyanogen bromide agarose (via covalent attachment) and on octyl agarose (via physical adsorption on the hydrophobic support by interfacial activation). Both immobilized preparations have been incubated in dextran sulfate (DS) or polyethylenimine (PEI) solutions to coat the enzyme surface. Then, the activity versus different substrates and under different experimental conditions was evaluated. The PEI coating generally produced a significant increase in enzyme activity, in some cases even by more than a 30-fold factor (using the octyl-Lecitase at pH 5 in the hydrolysis of methyl phenyl acetate). In opposition, the DS coating usually produced some negative effects on the enzyme activity. The rate of irreversible inhibition of the covalent preparation using diethyl p-nitrophenylphosphate did not increase after PEI coating suggesting that the increase in Lecitase activity is not a consequence of the stabilization of the open form of Lecitase. Moreover, the coating greatly increased the stability of the immobilized Lecitase, for example using DS and the covalent preparation, the half-life was increased by a 30-fold factor in 30% acetonitrile. The stabilizing effect was not found in all cases, in certain cases even a certain destabilization is found (e.g., octyl-Lecitase-DS at pH 7). Thus, the effects of the ionic polymer coating strongly depend on the substrate, experimental conditions and immobilization technique employed.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista.

The phospholipases A(1) (PLA(1) s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1) , combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2) s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Purification, sequencing and structural characterization of the phospholipase A1 from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae).

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.