Biochem. J. (2002) 364, 49–55 (Printed in Great Britain) 49 The same rat Δ6-desaturase not only acts on 18- but also on 24-carbon fatty acids in very-long-chain polyunsaturated fatty acid biosynthesis Sabine D’ANDREA*, Herve! GUILLOU*, Sophie JAN*†, Daniel CATHELINE*, Jean-Noe$ l THIBAULT‡, Monique BOURIEL*, Vincent RIOUX* and Philippe LEGRAND* 1 *Laboratoire de Biochimie, INRA-ENSA, 65 rue de Saint-Brieuc, CS84215, 35042 Rennes cedex, France, †Universite! du Maine, Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, France, and ‡UMR-VP INRA-ENSA, 35590 Saint-Gilles, France The recently cloned Δ6-desaturase is known to catalyse the first step in very-long-chain polyunsaturated fatty acid biosynthesis, i.e. the desaturation of linoleic and α-linolenic acids. The hypothesis that this enzyme could also catalyse the terminal desaturation step, i.e. the desaturation of 24-carbon highly unsaturated fatty acids, has never been elucidated. To test this hypothesis, the activity of rat Δ6-desaturase expressed in COS-7 cells was investigated. Recombinant Δ6-desaturase expression was analysed by Western blot, revealing a single band at 45 kDa. The putative involvement of this enzyme in the Δ6-desaturation of C #% : & nfi3 to C #% : ’ nfi3 was measured by incubating transfect- ed cells with C ## : & nfi3. Whereas both transfected and non- transfected COS-7 cells were able to synthesize C #% : & nfi3 by INTRODUCTION In animal cells, the availability of nfi6 and nfi3 very-long-chain polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA; C ## : ’ nfi3), depends both on the diet providing the precursors and on the activity of enzymes involved in very-long- chain PUFA biosynthesis [1]. The described pathway for DHA synthesis from α-linolenic acid involves alternating steps of desaturation and elongation [2]. The first part of the biosynthesis (Δ6-desaturation of α-linolenic acid, elongation, Δ5-desaturation and elongation) leading to C ## : & nfi3 has been well-documented [3,4]. The mammalian genes encoding Δ6- and Δ5-desaturases involved in the two initial desaturation steps have been identified [5–8]. However, the final conversion of C ## : & nfi3 to C ## : ’ nfi3 remains controversial. For years, the presence of a Δ4-desaturase has been assumed [9]. In 1991, a modified pathway independent of a Δ4-desaturase was proposed [10]. The pathway includes, successively, an elongation, a Δ6-desaturation and a final peroxi- somal β-oxidation [11,12], as follows : C ## : & nfi3!C #% : & nfi3!C #% : ’ nfi3!C ## : ’ nfi3. The hypothesis that the same Δ6-desaturase, without chain- length specificity, could act on both 18-carbon essential fatty acids and 24-carbon highly unsaturated fatty acids has never been tested. Competitive studies with rat liver microsomes indicated that a single Δ6-desaturase may act on both 18- and 24- carbon PUFA substrates [13]. Recent work based on the identi- fication of the first Δ6-desaturation-step deficiency in humans [14,15] has also suggested that a single Δ6-desaturase may be involved in the two different desaturations. However, metabolic Abbreviations used : DHA, docosahexaenoic acid ; FCS, foetal calf serum ; GST, glutathione S-transferase ; PUFA, polyunsaturated fatty acids ; DMEM, Dulbecco’s modified Eagle’s medium. 1 To whom correspondence should be addressed (e-mail Philippe.Legrand!agrorennes.educagri.fr). elongation of C ## : & nfi3, only cells expressing Δ6-desaturase were also able to produce C #% : ’ nfi3. In addition, Δ6-desaturation of [1-"%C]C #% : & nfi3 was assayed in itro in homogenates from COS-7 cells expressing Δ6-desaturase or not, showing that Δ6- desaturase catalyses the conversion of C #% : & nfi3 to C #% : ’ nfi3. Evidence is therefore presented that the same rat Δ6-desaturase catalyses not only the conversion of C ") : $ nfi3 to C ") : % nfi3, but also the conversion of C #% : & nfi3 to C #% : ’ nfi3. A similar mechanism in the nfi6 series is strongly suggested. Key words : docosahexaenoic acid, nfi3 fatty acid, α-linolenic acid, tetracosahexaenoic acid. studies based on different conversion rates have suggested that there may also be two different Δ6-desaturase isoforms [16,17]. These studies support the existence of distinct enzymes catalysing Δ6-desaturation of 18- and 24-carbon fatty acids. In the present work, the hypothesis that the protein encoded by the rat liver Δ6-desaturase cDNA [6] could act on both 18- and 24-carbon essential fatty acids was investigated. Evidence is presented that recombinant rat Δ6-desaturase expressed in mam- malian cells catalyses not only the conversion of C ") : $ nfi3 to C ") : % nfi3, but also the conversion of C #% : & nfi3 to C #% : ’ nfi3. MATERIALS AND METHODS Chemicals cis-7,10,13,16,19-Docosapentaenoic acid (C ## : & nfi3) and cis- 9,12,15,17,21-tetracosapentaenoic acid (C #% : & nfi3) were pur- chased from Matreya (Pleasant Gap, PA, U.S.A.) and American Radiolabelled Chemicals (St. Louis, MO, U.S.A.), respectively. The characterized fatty acid methyl ester of C #% : ’ nfi3 [18] was a gift from Dr K. Ishihara (National Research Institute of Fisheries Science, Yokohama, Japan). Other unlabelled fatty acids were from Sigma (St Quentin Fallavier, France). Radio- labelled [1-"%C]C ") : $ nfi3 (52 mCi}mmol) and [1-"%C]C #% : & nfi3 (55 mCi}mmol) were purchased from NEN Life Science (Paris, France) and American Radiolabelled Chemicals respectively. Foetal calf serum (FCS) was purchased from Perbio (Bezons, France). Solvents (HPLC grade) were purchased from Fischer # 2002 Biochemical Society