CANAVOSO LILIAN ETELVINA
Congresos y reuniones científicas
Título:
The B chain of ATP synthase is a lipophorin binding protein involved in lipid transfer in the midgut of Panstrongylus megistus (Hemiptera: Reduviidae)
Autor/es:
FRUTTERO LL; ARRESE, EL; CARLINI, C.R.; SOULAGES JL; RUBIOLO ER; CANAVOSO LE
Lugar:
Amsterdam
Reunión:
Simposio; Seventh International Symposium on Molecular Insect Science; 2014
Institución organizadora:
Center for Insect Science
Resumen:
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 tandem
mass spectrometry to identify the β chain of ATP synthase (β-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 β-
ATPase nucleotide sequence of Rhodnius prolixus, a related hemipteran species. As shown by
RT-PCR, the expression of the β-ATPase mRNA changed according to the nutritional status of
the insect. Moreover, the β-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 β-ATPase in lipid transfer was further assessed at
biochemical and cellular levels. β-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 β-ATPase partially co-localized with lipophorin, supporting the binding of these
proteins. The interaction of β-ATPase and the endogenous lipophorin was evidenced by
immunoprecipitation assays using microsomal fractions of the midgut. In vivo functional studies
showed 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
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 tandem
mass spectrometry to identify the β chain of ATP synthase (β-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 β-
ATPase nucleotide sequence of Rhodnius prolixus, a related hemipteran species. As shown by
RT-PCR, the expression of the β-ATPase mRNA changed according to the nutritional status of
the insect. Moreover, the β-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 β-ATPase in lipid transfer was further assessed at
biochemical and cellular levels. β-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 β-ATPase partially co-localized with lipophorin, supporting the binding of these
proteins. The interaction of β-ATPase and the endogenous lipophorin was evidenced by
immunoprecipitation assays using microsomal fractions of the midgut. In vivo functional studies
showed 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
