The B chain of ATP synthase is a lipophorin binding protein involved in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)

FRUTTERO LL; ARRESE, EL; CARLINI, C.R.; SOULAGES JL; RUBIOLO ER; CANAVOSO LE

Amsterdam

Simposio; Seventh International Symposium on Molecular Insect Science; 2014

Center for Insect Science

Lipophorin is the main lipid carrier in the hemolymph of the insects. Lipophorin functions as areusable shuttle, cycling among the target tissues by loading and unloading its lipid cargowithout synthesis or degradation of its apolipoprotein matrix. Currently, there are fewcharacterized candidates supporting the functioning of the docking mechanism of lipophorin mediated lipid transfer. In this work, we employed a combination of ligand blotting and tandemmass spectrometry to identify the β chain of ATP synthase (β-ATPase) as a lipophorin bindingprotein in the midgut membrane of the blood sucking insect Panstrongylus megistus, animportant Chagas´ disease vector in South America. Primers were designed based on thepartial amino acid sequence of peptides obtained from mass spectrometry and from the β-ATPase nucleotide sequence of Rhodnius prolixus, a related hemipteran species. As shown byRT-PCR, the expression of the β-ATPase mRNA changed according to the nutritional status ofthe insect. Moreover, the β-ATPase was cloned, sequenced and expressed in a heterologoussystem and the full-length transcript presented 1563 nucleotides and a deduced primarysequence of 521 amino acids. The role of β-ATPase in lipid transfer was further assessed atbiochemical and cellular levels. β-ATPase was detected by western blot in enriched midgutmembrane preparations free of mitochondria. Its localization in the plasma membrane ofenterocytes was confirmed by immunofluorescence using isolated enterocytes. It was alsoshown that β-ATPase partially co-localized with lipophorin, supporting the binding of theseproteins. The interaction of β-ATPase and the endogenous lipophorin was evidenced byimmunoprecipitation assays using microsomal fractions of the midgut. In vivo functional studiesshowed that blocking β-ATPase impaired lipophorin binding to midgut tissue and lipophorin mediated lipid transfer to midgut cells. Taken together, the findings strongly suggest that β-ATPase plays a role as a docking lipophorin receptor in the midgut of P. megistus.Keywords: Triatominae, midgut, lipophorin receptors, β chain of ATP synthase