PLASMEPSIN INHIBITORS; IS PLA2 ENZYME THE NATURAL INHIBITOR IN HUMANS AGAINST PLASMEPSINS PRODUCED BY MALARIAL PARASITE IN THEIR ERYTHROCYTIC CYCLE?

Malaria is a mosquito-borne infectious disease that affects humans and other animals. Most deaths in malarial infection are caused by P. falciparum because P. vivax, P. ovale, and P. malariae generally cause a milder form of malaria. Within the red blood cells, the parasites multiply further, again asexually, periodically breaking out of their host cells to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cells. Plasmepsin is a hemoglobin-degrading enzyme produced by the plasmodium parasite. It is an aspartic acid protease having 2 aspartic acid residues in the active site. On the other hand phospholipase A2 levels are increased in malarial infection and this may possibly provide protection against the effects of plasmepsin. This review examines the importance of this enzyme and interaction with plasmepsin.

Three Dangerous Loops Of Lipoproteine-Associated Phospholipase A2 Activity On Increasing LDL Aterogenecity

Background. Hypercholesterolemia is a major classic risk factor for cardiovascular disease, but there are 35%-40% cases of cardiovascular patients have a normal cholesterol levels. Lp-PLA2 is an enzyme that produced and secreted by macrophages as a response to the lipid peroxide formation, especially the platelet activating factor compound and phosphocholine peroxide. Lp-PLA2 is correlated with classic risk factor of cardiovascular disease, although that correlation with number of foam cell at early stage of atherosclerosis is not clear yet. This study aims to determine whether Lp-PLA2 levels correlated with classic risk factors of atherosclerosis and the number of foam cell, and the role of Lp-PLA2 enzyme in foam cells formation.Methods. This study observes the change of Lp-PLA2, F2-Isp, MDA, TC, LDL, HDL levels in rat serum at 3 levels of early atherogenesis, Ath-I, Ath-II and Ath-III were made on the number of foam cells. The number of cells was observed on all aortic cross sectional surfaces, using the Oil-Red-O staining. The LDL-C content was measure using the Fiedwall formula, MDA content was measure by using TBA-test, the observe of F2-isoprostane and Lp-PLA2 followed the procedure Elisa methods. Results. Anova test results among the 3 initial atherosclerotic levels showed a very significant difference (p<0.01) on Lp-PLA2 plasma content. The LSD test results represented an increase in Lp-PLA2 enzyme levels significantly since AthII stage. Path analysis refers that correlation value between the Lp-PLA2and the number of foam cell (r=0.48) were higher than that of the LDL (r=0.42), was neither correlated with MDA nor F2-Isp, the highest correlation occurred between Lp-PLA2 and LDL compared to the others parameters (r = 0.58). Path analysis also showed no correlation between cell numbers with MDA and F2-Isp, but LDL levels are correlated significantly with of oxidative stress markers MDA levels (r = 0.32) and correlated very significantly with F2-Isp (r = 0.69).Conclussion. It can be concluded that elevated levels of Lp-PLA2 increase atherogenecity of LDL, due to increased inflammation, stress oxidation and elevated levels of Lp-PLA2 itself, wich are interconnected with proatherogenic loops.

Adeno-Associated Virus VP1u Exhibits Protease Activity

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

Antisnake venom activity and isolation of quercetin from the leaf of Neocarya macrophylla (Sabine) Prance ex F. White (Malpighiales: Chrysobalanaceae)

Snake envenomation is a major cause of death and disability in many developing countries. Neocarya macrophylla (Sabine) Prance ex F. White (Malpighiales: Chrysobalanaceae) have been reportedly used in traditional medicine to treat snake envenomation. Bioassay-guided isolation of antivenom principles was carried out on the leaf of N. macrophylla against Naja nigricollis venom. The methanol extract of N. macrophylla leaf and its ethylacetate and n-butanol fraction significantly (P < 0.05) protected mice against venom-induced lethality with 100% survival rate and there was remarkable inhibition of the poisonous effects of PLA2 enzyme by the extracts and the fractions. Encouraged by this result, the ethylacetate soluble fraction was subjected to purification using vacuum liquid chromatography and gel filtration which led to the isolation of quercetin as the bioactive principle. The identity of the compound was determined on the basis of chemical tests, and by comparison of its 1H-NMR data with literature, this is the first report of isolation of this compound from the leaf of the plant. However, the results of the study suggests that the leaf of N. macrophylla possess significant antisnake venom activity which provide the scientific basis for its use in traditional treatment of snakebites.

Elevated circulating levels of lipoprotein-associated phospholipase A2 in obese children

Obesity and cardiovascular disease (CVD) often co-exist, but the pathophysiologic mechanisms that link the two are not fully understood. Lipoprotein-associated phospholipase ASixty-seven lean [39 boys and 28 girls, mean body mass index (BMI) z-score –0.2±0.8] and 66 obese (32 boys and 34 girls, mean BMI z-score 4.4±1.2) age-matched (p=0.251) children, aged 6–12 years, were studied. BMI z-score was calculated based on the Greek BMI growth curves, and children were categorized as obese according to the Cole criteria. All children underwent physical examination and a fasting morning blood sample was obtained for glucose, insulin, lipid profile, and Lp-PLA2 assessment. Plasma concentrations of Lp-PLA2 were determined by a commercially available Lp-PLA2 enzyme-linked immunosorbent assay kit (PLAC Test), while other measurements were performed using standard methods.Plasma Lp-PLA2 levels were significantly higher in obese children (322.5±77.8 ng/mL) compared with normal-weight ones (278.0±64.4 ng/mL, p<0.001). Lp-PLA2 concentrations were significantly correlated with the BMI z-score (p=0.004). Receiver operating characteristic analysis on Lp-PLA2 values resulted in significant areas under the curve (AUC) for distinguishing between obese and normal-weight groups of children (AUC, 0.726; p<0.001).We found significantly higher Lp-PLA2 levels in obese children than lean controls. Interestingly, they all had levels >200 ng/mL, which are considered to correlate with atherosclerosis and a high thromboembolic risk in adults. The positive correlation of Lp-PLA2 with BMI suggests that Lp-PLA2 might be the link between obesity and increased cardiovascular risk, which can be elevated even at a very young age. Measurement of Lp-PLA2 in plasma could therefore represent a further biomarker for assessing increased CVD risk in obese children and adolescents.

Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.