Galactosylceramide synthase of Trypanosoma cruzi: purification and characterization

J. E. PARENTE; M. LANDONI; V. DUSCHAK; A. S. COUTO

Villa Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2008

Ceramide UDP-GalT [E.C.: 2.4.1.45] is a key enzyme in the biosynthesis of cerebrosides catalyzing the transfer of Galactose from UDP-Galactose to Ceramide yielding Galactosylceramide (GalCer). This glycosphingolipid serves as a precursor for a few simple glycolipids, sulfatide, galabiosylceramide, and the ganglioside sialo-Galceramide, however Galactosylceramide is the major constituent of myelin where the Ceramide UDP-GalT activity changes during the myelination process. It is also highly enriched in many epithelial cells where it is thought to play an important role in lipid and protein sorting. Although the main pathways for glycosphingolipid biosynthesis are relatively well understood in mammalian cells, little is known about them in parasites.              In the present work we describe the purification and partial characterization of the Ceramide UDP-GalT obtained from epimastigote forms of Trypanosoma cruzi. The enzyme activity was determined in total lysates obtained from Tul-2 and CL-Brenner strains as well as semipurified membrane fractions. Fractionation on ConA-Sepharose column yielded a protein band with an apparent MW range of 62-68 kDa whose activity was shown using UDP-Gal and NBD-Ceramide as sustrates. On going studies of Maldi mass spectrometry analysis to determine the sequence of the purified protein and biochemical characterization will be described.