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

FRUTTERO, L.L.; ARRESE, E.L.; CARLINI, C.R.; SOULAGES, J.L.; RUBIOLO, E.R.; CANAVOSO, L.E.

Amsterdan

Simposio; Seventh International Symposium on Molecular Insect Science; 2014

Elsevier and Center for Insect Science (University of Arizona)

Lipophorin is the main lipid carrier in the hemolymph of the insects. Lipophorin functions as a reusable shuttle, cycling among the target tissues by loading and unloading its lipid cargo without synthesis or degradation of its apolipoprotein matrix. Currently, there are few characterized candidates supporting the functioning of the docking mechanism of lipophorin-mediated lipid transfer. In this work, we employed a combination of ligand blotting and tanden mass spectrometry to identify the Beta chain of ATP synthase (Beta-ATPase) as a lipophorin binding protein in the midgut membrane of the blood sucking insect Panstrongylus megistus, an important Chagas' disease vector in South America. Primers were designed based on the partial amino acid sequence of peptides obtained from mass spectrometry and from the Beta-ATPase nucleotide sequence of Rhodnius prolixus, a related hemipteran species. As shown by RT-PCR, the expression of the Beta-ATPase mRNA changed according to the nutritional status of the insect. Moreover, the Beta-ATPase was cloned, sequenced and expressed in a heterologous system and the full-length transcript presented 1563 nucleotides and a deduced primary sequence of 521 amino acids. The role of Beta-ATPase in lipid transfer was further assessed at biochemical and cellular levels. Beta-ATPase was detected by western blot in enriched midgut membrane preparations free of mitochondria. Its localization in the plasma membrane of enterocytes was confirmed by immunofluorescence using isolated enterocytes. It was also shown that Beta-ATPase partially co-localized with lipophorin, supporting the binding of these proteins. The interaction of Beta-ATPase and the endogenous lipophorin was evidenced by immunoprecipitation assays using microsomal fractions of the midgut. In vivo functional studies showed that blocking Beta-ATPase impaired lipophorin binding to midgut tissue and lipophorin-mediated lipid transfer to midgut cells. Taken together, the findings strongly suggest that Beta-ATPase plays a role as a docking lipophorin receptor in the midgut of P. megistus.