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

VACCHINA P.; ROSSO C.; TRIPODI K.; UTTARO A.

Villa Carlos Paz. Córdoba. Argentina

Congreso; XLIV Reunio Anual- 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 trytrip 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 scarcely related with fungi’s group, in which a few enzymes have desaturase/ hydroxylase activity.Percentages of identities of T. brucei protein were 53.9, 50- 51.9 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). In both cases it was possible to detect desaturase but no hydroxylase activity, reason why we classify them as D4-SphDes. Epimastigotes of T. cruzi showed mainly 18C DHS, followed by C18 Sph, low level of 20C DHS and no PSph. Since some reports depict the presence of low levels of PSph in L. major, we are currently investigating these enzymes, as some of them might be hydroxylases or bifunctional enzymes.