Cloning and functional characterization of Delta 4- sphingolipid desaturases from trypanosomatids

VACCHINA, PAOLA; ROSSO, COSTANZA; UTTARO, ANTONIO D.; TRIPODI, KARINA

Villa Carlos Paz, Córdoba, Argentina

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

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Sphingolipids (Spl) are ubiquitous compounds present in membrane of eukaryotic cells. They comprise a long chain base (LCB) with and amide linked fatty acid. C1 OH may be substituted and LCB may be saturated (Dihydrosphingosine, DHS), unsaturated (Sphingosine, Sph) or hydroxylated (Phytosphingosine, PSph) in position 4. This desaturation is carried out by a D4-SphDes. In Tritryp database, we found 3 sequences in Trypanosoma cruzi (1 is entire), 1 in T. brucei and 4 in Leishmania major (3 are identical). A phylogenetic analysis unveiled that trypanosomatid proteins define a cluster related with fungi’s group, where a few enzymes have desaturase/ hydroxylase activity. Percentages of identities of T. brucei protein are 54, 52 and 37% to the enzymes of T. cruzi, L. major and Schyzosaccaromyces pombe (a D4-SphDes), respectively. We cloned the enzymes of T. brucei and T. cruzi in pYES2, and analyzed the enzymatic activity in a yeast mutant for LCB hydroxylase (sur2). We detected desaturase but no hydroxylase activity for the enzyme of T. brucei, reason why we classify it as D4-SphDes. T. cruzi and T. brucei cultures showed mainly 18C DHS, followed by C18 Sph, low level of 20C and 16C DHS, and no PSph. Since some reports depict the presence of minimal levels of PSph in L. major, we are currently investigating these enzymes, as some of them might be hydroxylases or bifunctional enzymes.