Genome analysis of sphingolipid metabolism related genes in Tetrahymena thermophila andidentification of a Fatty Acid 2 Hydroxylase gene by somatic knockout

NICOLAS G. CID, MARIA L. SANCHEZ GRANEL, CLARA B. NUDEL, ALEJANDRO D. NUSBLAT

Camerino

Conferencia; CILIATE MOLECULAR BIOLOGY 2015; 2015

Universita de Camerino

Genome analysis of sphingolipid metabolism related genes in Tetrahymena thermophila andidentification of a Fatty Acid 2 Hydroxylase gene by somatic knockout. Nicolas G. Cid, Maria L. Sanchez Granel, Clara B. Nudel, Alejandro D. Nusblat. CILIATE MOLECULAR BIOLOGY 2015, JULY 10th to 16th 2015, CAMERINO, ITALY. Sphingolipids along with glycerophospholipids and sterols are the primary structural lipids present in membranes of eukaryotic cells, and as other lipids alike, they play additional roles as second messengers in regulation of signal transduction pathways. They comprise a diverse group of over 100 bioactive different moieties involved in fundamental cellular processes, such as proliferation, survival, cell death, adhesion, migration, angiogenesis, and embryogenesis. The lipid composition of the functionally different membranes, including glycerophospholipids, sterols and sphingolipids, has been studied extensively in ciliates such as Tetrahymena and Paramecium. Nevertheless no related gene involved in sphingolipid metabolism has been identified so far. To address this issue we have undertaken a genome-wide survey of putative genes involved in sphingolipid metabolism in Tetrahymena thermophila from which a number of candidates have been selected and we have also identified a sphingolipid fatty acid 2-hydroxylase in the ciliate by somatic knockout. This enzyme belongs to the fatty acid hydroxylase superfamily, and catalyzes the hydroxylation at position 2 of fatty acids that will then be attached to sphingoid bases, forming ceramides with N-acyl hydroxy fatty acid chain. A gene encoding for this same enzyme was first identified in the yeast Saccharomyces cerevisiae and later in mammals (Homo sapiens, Mus musculus and Rattus norvegicus) and plant (Arabidopsis thaliana). Although the physiological role of cell and 2-HAG is largely unknown, in S. cerevisiae 2-OH sphingolipids are involved in endocytosis, while its alteration is associated to neurodegenerative diseases in humans. Gene deletion of the only fatty acid 2-hydroxylase ortholog present in T. thermophila (TTHERM_00463850) eliminates the ability to synthesize sphingolipids with hydroxylations in the fatty acid chain. Gas chromatography coupled mass spectrometry analysis of purified sphingolipids, showed that main 2-HFAs (16h:0, 17h:0, 18h:0 and 19h:0) present in the wild type strain were unable to be detected in the knockout strain. The activity is not vital to the cell, though it may be linked to other phenotypes preliminarly assayed Further studies of sphingolipid metabolism in ciliates could contribute to the understanding of the cellular and molecular mechanisms of metabolic pathways not only in the ciliates but also in different human pathologies associated with sphingolipids disorders, such as Gaucher´s disease, Niemann-Pick, among others.