CALCIUM-INDUCED LIPID PEROXIDATION IS MEDIATED BY RHODNIUS HEME-BINDING PROTEIN (RHBP) AND PREVENTED BY VITELLIN.

Lipid peroxidation is promoted by the quasi-lipoxygenase (QL) activity of heme proteins and enhanced by the presence of free calcium. Unlike mammalian plasma, the hemolymph of Rhodnius prolixus, a vector of Chagas disease, contains both a free heme-binding protein (RHBP) and circulating lipoproteins. RHBP binds and prevents the heme groups of the proteins from participating in lipid peroxidation reactions. Herein, we show that despite being bound to RHBP, heme groups promote lipid peroxidation through a calcium-dependent QL reaction. This reaction is readily inhibited by the presence of ethylene glycol tetraacetic acid (EGTA), the antioxidant butylated hydroxytoluene or micromolar levels of the main yolk phosphoprotein vitellin (Vt). The inhibition of lipid peroxidation is eliminated by the in vitro dephosphorylation of Vt, indicating that this reaction depends on the interaction of free calcium ions with negatively charged phosphoamino acids. Our results demonstrate that calcium chelation mediated by phosphoproteins occurs via an antioxidant mechanism that protects living organisms from lipid peroxidation.

Tick vitellin is dephosphorylated by a protein tyrosine phosphatase during egg development: effect of dephosphorylation on VT proteolysis.

Vitellin (VT) is a phospholipoglycoprotein that is the main component of arthropod egg yolk. Phosphorylation is a recurrent feature of every VT molecule described so far. However, the role played by such post-translational modification during egg development is not yet clear. In the eggs of the hard tick Boophilus microplus, VT is a phosphotyrosine-containing protein. VT-phosphotyrosine residues are gradually removed during tick embryogenesis due to the action of a 45 kDa egg tyrosine phosphatase. This enzyme is strongly inhibited by ammonium molybdate, sodium vanadate and cupric ion. The role of phosphotyrosine residues in VT proteolytic degradation was evaluated. Western blots probed with a monoclonal anti-phosphotyrosine antibody demonstrated that the high molecular mass VT subunits (VT 1 and VT 2) are the main targets of dephosphorylation during egg development. Both dephosphorylation and proteolysis of VT 1 and VT 2 are blocked by ammonium molybdate in total egg homogenates. When purified VT was dephosphorylated in vitro with lambda phosphatase and then incubated in the presence of bovine cathepsin D, VT proteolysis increased dramatically. Altogether, these data are the first to show that phosphotyrosine residues are present in a yolk protein, and that such residues might be involved in the regulation of VT breakdown during egg development.

Oocyte fertilization triggers acid phosphatase activity during Rhodnius prolixus embryogenesis.

Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na(+)/K(+) tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of (32)P from (32)P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.