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Proteomic Approach for Characterization of Immunodominant Membrane-Associated 30- to 36-Kilodalton Fraction Antigens of Leishmania infantum Promastigotes, Reacting with Sera from Mediterranean Visceral Leishmaniasis Patients

Laboratoire d'Immunologie, Vaccinologie et Génétique Moléculaire,¹ Laboratoire d'Epidémiologie et d'Ecologie Parasitaire, Institut Pasteur de Tunis,² Laboratoire de Biochimie Végétale et Symbiotes, Institut National de la Recherche Scientifique et Technique, Hammam-Lif, Tunis, Tunisia,⁵ Laboratoire de Spectrométrie de Masse Bio-Organique (UMR 7509), Strasbourg,³ Laboratoire de Biochimie CNRS (UMR 6560 and LIA Ingénierie Biomoléculaire), Faculté de Médecine Nord, Marseille, France⁴

Received 15 January 2004/ Returned for modification 4 May 2004/ Accepted 6 October 2004

The aim of the present study was to identify and characterize proteins of a 30- to 36-kDa fraction of Leishmania infantum promastigote membranes previously shown to be an immunodominant antigen(s) in Mediterranean visceral leishmaniasis (MVL) and a consistent and reliable serological marker of this disease. By the first approach, Coomassie-stained protein bands (32- and 33-kDa fractions) that specifically reacted by immunoblotting with sera from MVL patients were excised from the gel and submitted to enzymatic digestion to generate peptides. Four peptides were sequenced, three of which were shown to be definitely associated with MVL-reactive antigens and ascribed to a mitochondrial integral ADP-ATP carrier protein from L. major, a putative NADH cytochrome b₅ reductase, and a putative mitochondrial carrier protein, respectively. The second approach combined two-dimensional gel electrophoresis of membrane antigens and mass spectrometry (liquid chromatography-mass spectrometry/mass spectrometry) by using a quadrupole time-of-flight analysis. Six immunoreactive spots that resolved within a molecular mass range of 30 to 36 kDa and a pH range of 6.7 to 7.4 corresponded to four Leishmania products. The sequences derived from two spots were ascribed to a beta subunit-like guanine nucleotide binding protein, known as the activated protein kinase C receptor homolog antigen LACK, and to a probable member of the aldehyde reductase family. One spot was identified as a probable ubiquinol-cytochrome c reductase (EC 1.10.2.2) Rieske iron-sulfur protein precursor. The remaining three spots were identified as truncated forms of elongation factor 1. These antigens correspond to conserved proteins ubiquitously expressed in eukaryotic cells and represent potential candidates for the design of a reliable tool for the diagnosis of this disease.