Molecular and functional characterization of a SCD 1b from European sea bass (Dicentrarchus labrax L.)

Abstract

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5 kb and 1.4 kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Δ9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of α-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe²⁺ ions. A new His-containing motif NX₃H-like includes an Asn residue that participates in the coordination of Fe²⁺1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Δ gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Δ6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Δ6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish.

Keywords: 3D protein structure; Dicentrarchus labrax L.; Functional characterization; MUFAs; Molecular cloning; SCD 1b; Substrate preference; Substrate specificity; Δ6 FAD.