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Originally published In Press as doi:10.1194/jlr.M600285-JLR200 on July 22, 2006

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Journal of Lipid Research, Vol. 47, 2171-2178, October 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Characterization of Leishmania (Viannia) braziliensis membrane microdomains, and their role in macrophage infectivity

Kelly A. G. Yoneyama*, Ameria K. Tanaka*, Thais G. V. Silveira{dagger}, Helio K. Takahashi* and Anita H. Straus*,1

* Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo, SP 04023-900, Brazil
{dagger} Departamento de Análises Clínicas, Universidade Estadual de Maringá, Maringá, PR 87020-900, Brazil

Published, JLR Papers in Press, July 22, 2006.

1 To whom correspondence should be addressed. e-mail: straus.bioq{at}epm.br

Detergent-resistant membranes (DRMs) from Leishmania (Viannia) braziliensis promastigotes, insoluble in 1% Triton X-100 at 4°C, were fractionated by sucrose density gradient ultracentrifugation. They were composed of glycoinositolphospholipids (GIPLs), inositol phosphorylceramide (IPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sterols. In contrast, 1% Triton X-100-soluble fraction was composed of PE, phosphatidylcholine, phosphatidylserine, PI, IPC, sterol, and lyso-PI. High-performance thin-layer chromatography (HPTLC) immunostaining using monoclonal antibody SST-1 showed that 85% of GIPLs are present in DRMs, and immunoelectron microscopic analysis showed that SST-1-reactive components are located in patches along the parasite surface. No difference in GIPL pattern was observed by HPTLC between Triton X-100-soluble versus -insoluble fractions at 4°C. Analysis of fatty acid composition in DRMs by GC-MS showed the presence of GIPLs containing an alkylacylglycerol, presenting mainly saturated acyl and alkyl chains. DRMs also contained sterol, IPC with saturated fatty acids, PI with at least one saturated acyl chain, and PE with predominantly oleic acid. Promastigotes treated with methyl-ß-cyclodextrin to disrupt lipid microdomains showed significantly lower macrophage infectivity, suggesting a relationship between lipid microdomains and the infectivity of these parasites.

Supplementary key words glycolipid • inositol phosphorylceramide • lipid raft • promastigote • detergent-resistant membranes • gas chromatography-mass spectrometry • methyl-ß-cyclodextrin

Abbreviations: AGPB, phosphate buffer containing 0.5% bovine serum albumin and 0.1% gelatin; DRM, detergent-resistant membrane; FAME, fatty acid methyl ester; GIPL, glycoinositolphospholipid; GPI, glycosylphosphatidylinositol; HPTLC, high-performance thin-layer chromatography; IPC, inositol phosphorylceramide; MAb, monoclonal antibody; MßCD, methyl-ß-cyclodextrin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine


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