RESUMO
The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.
Assuntos
Ácidos Graxos Insaturados/metabolismo , Proteínas Fúngicas/genética , Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Acetiltransferases , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of gamma-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two Delta6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina Delta5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.
Assuntos
Acetiltransferases/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Mortierella/enzimologia , Ácido gama-Linolênico/biossíntese , Acetiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Elongases de Ácidos Graxos , Dados de Sequência Molecular , Mortierella/genética , Mortierella/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Two human expressed sequence tag (EST) cDNA sequences with identity with Delta(5)- and Delta(6)-desaturases from a filamentous fungus, Mortierella alpina, were identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. (Palo Alto, CA, U.S.A.). An oligonucleotide complementary to the 3' EST cDNA sequences was used to screen human liver cDNA using rapid amplification of cDNA ends (RACE)-PCR. The amplified DNA fragment had 98% identity with a putative open reading frame (ORF) predicted from a human genomic sequence, and encoded 444 amino acids. Expression of this ORF in mouse fibroblast cells demonstrated that the encoded protein was a Delta(5)-desaturase, as determined by the conversion of dihomo-gamma-linolenic acid (C(20:3,n-6)) into arachidonic acid (C(20:4,n-6)). The human Delta(5)-desaturase contained a predicted N-terminal cytochrome b(5)-like domain, as well as three histidine-rich domains. A tissue expression profile revealed that this gene is highly expressed in fetal liver, fetal brain, adult brain and adrenal gland. A search of the existing databases led to localization of this ORF within a 14 kb interval flanked by the flap endonuclease-1 (FEN1) and vitelliform macular dystrophy (Best's disease; VMD2) loci of chromosome 11q12.
Assuntos
Ácido Araquidônico/biossíntese , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Sequência de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Cromossomos Humanos Par 11/genética , Clonagem Molecular , DNA Complementar/genética , Bases de Dados Factuais , Dessaturase de Ácido Graxo Delta-5 , Etiquetas de Sequências Expressas , Ácidos Graxos Dessaturases/química , Ácidos Graxos/análise , Perfilação da Expressão Gênica , Humanos , Células L , Linoleoil-CoA Desaturase , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Mapeamento Físico do Cromossomo , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Transfecção , Ácido gama-Linolênico/metabolismoRESUMO
We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.
