RESUMEN
The splendid alfonsino Beryx splendens is a commercially important deep-sea fish in East Asian countries. Because the wild stock of this species has been declining, there is an urgent need to develop aquaculture systems. In the present study, we investigated the long-chain polyunsaturated fatty acid (LC-PUFA) requirements of B. splendens, which are known as essential dietary components in many carnivorous marine fish species. The fatty acid profiles of the muscles, liver, and stomach contents of B. splendens suggested that it acquires substantial levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from its natural diet. The functional characterization of a fatty acid desaturase (Fads2) and three elongases (Elovl5, Elovl4a, and Elovl4b) from B. splendens confirmed their enzymatic capabilities in LC-PUFA biosynthesis. Fads2 showed Δ6 and Δ8 bifunctional desaturase activities. Elovl5 showed preferential elongase activities toward C18 and C20 PUFA substrates, whereas Elovl4a and Elovl4b showed activities toward various C18-22 substrates. Given that Fads2 showed no Δ5 desaturase activity and no other fads-like sequence was found in the B. splendens genome, EPA and arachidonic acid cannot be synthesized from C18 precursors; hence, they can be categorized as dietary essential fatty acids in B. splendens. EPA can be converted into DHA in B. splendens via the so-called Sprecher pathway. However, given that fads2 is only expressed in the brain, it is unlikely that the capacity of B. splendens to biosynthesize DHA from EPA can fulfill its physiological requirements. These results will be useful to researchers developing B. splendens aquaculture methods.
Asunto(s)
Proteínas de Peces , Peces , Animales , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Proteínas de Peces/metabolismo , Peces/metabolismo , Ácido Graso Desaturasas/genética , Ácidos Grasos Esenciales , Ácido Eicosapentaenoico , Ácidos Docosahexaenoicos , Dieta/veterinaria , Ácidos GrasosRESUMEN
Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a "Sprecher" pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid ß-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the "Sprecher" pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish.
Asunto(s)
Enoil-CoA Hidratasa , Pez Cebra , Animales , Enzima Bifuncional Peroxisomal/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Enoil-CoA Hidratasa/genética , Enoil-CoA Hidratasa/metabolismo , Enoil-CoA Hidratasa/farmacología , Peroxisomas/metabolismo , Hígado/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , 3-Hidroxiacil-CoA Deshidrogenasas/farmacología , Acetiltransferasas/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
There is a growing interest to understand the capacity of farmed fish species to biosynthesise the physiologically important long-chain (≥C20) n-3 and n-6 polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ARA), from their C18 PUFA precursors available in the diet. In fish, the LC-PUFA biosynthesis pathways involve sequential desaturation and elongation reactions from α-linolenic acid (ALA) and linoleic acid (LA), catalysed by fatty acyl desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) proteins. Our current understanding of the grass carp (Ctenopharyngodon idella) LC-PUFA biosynthetic capacity is limited despite representing the most farmed finfish produced worldwide. To address this knowledge gap, this study first aimed at characterising molecularly and functionally three genes (fads2, elovl5 and elovl2) with putative roles in LC-PUFA biosynthesis. Using an in vitro yeast-based system, we found that grass carp Fads2 possesses ∆8 and ∆5 desaturase activities, with ∆6 ability to desaturase not only the C18 PUFA precursors (ALA and LA) but also 24:5n-3 to 24:6n-3, a key intermediate to obtain DHA through the "Sprecher pathway". Additionally, the Elovl5 showed capacity to elongate C18 and C20 PUFA substrates, whereas Elovl2 was more active over C20 and C22. Collectively, the molecular cloning and functional characterisation of fads2, elovl5 and elovl2 demonstrated that the grass carp has all the enzymatic activities required to obtain ARA, EPA and DHA from LA and ALA. Importantly, the hepatocytes incubated with radiolabelled fatty acids confirmed the yeast-based results and demonstrated that these enzymes are functionally active.
Asunto(s)
Carpas , Ácido Graso Desaturasas , Ácidos Grasos Insaturados , Animales , Carpas/genética , Carpas/metabolismo , Ácidos Docosahexaenoicos/biosíntesis , Ácido Eicosapentaenoico/biosíntesis , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados/biosíntesis , Saccharomyces cerevisiaeRESUMEN
Sinocyclocheilus represents a rare, freshwater teleost genus endemic to China that comprises the river-dwelling surface fish and the cave-dwelling cavefish. Using a combinatorial approach of quantitative lipidomics and mass-spectrometry imaging (MSI), we demonstrated that neural compartmentalization of lipid distribution and lipid metabolism is associated with the evolution of troglomorphic traits in Sinocyclocheilus. Attenuated docosahexaenoic acid (DHA) biosynthesis via the Δ4 desaturase pathway led to reductions in DHA-phospholipids in cavefish cerebellum. Instead, cavefish accumulates arachidonic acid-phospholipids that may disfavor retinotectal arbor growth. Importantly, MSI of sulfatides coupled with immunostaining of myelin basic protein and transmission electron microscopy images of hindbrain axons revealed demyelination in cavefish raphe serotonergic neurons. Demyelination in cavefish parallels the loss of neuroplasticity governing social behavior such as aggressive dominance. Outside the brain, quantitative lipidomics and qRT-PCR revealed systemic reductions in membrane esterified DHAs in the liver, attributed to suppression of genes along the Sprecher pathway (elovl2, elovl5, and acox1). Development of fatty livers was observed in cavefish; likely mediated by an impeded mobilization of storage lipids, as evident in the diminished expressions of pnpla2, lipea, lipeb, dagla, and mgll; and suppressed ß-oxidation of fatty acyls via both mitochondria and peroxisomes as reflected in the reduced expressions of cpt1ab, hadhaa, cpt2, decr1, and acox1. These neurological and systemic metabolic adaptations serve to reduce energy expenditure, forming the basis of recessive evolution that eliminates nonessential morphological and behavioral traits and giving cavefish a selective advantage to thrive in caves where proper resource allocation becomes a major determinant of survival.
Asunto(s)
Characidae , Cyprinidae , Enfermedades Desmielinizantes , Animales , Evolución Biológica , Cuevas , Characidae/genética , Lipidómica , Redes y Vías Metabólicas , FosfolípidosRESUMEN
Long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA) are physiologically important nutrients for vertebrates including fish. Previous studies have addressed the metabolism of LC-PUFA in the Amazonian teleost tambaqui (Colossoma macropomum), an emerging species in Brazilian aquaculture, showing that all the desaturase and elongase activities required to convert C18 polyunsaturated fatty acids (PUFA) into LC-PUFA are present in tambaqui. Yet, elongation of very long-chain fatty acid 4 (Elovl4) proteins, which participate in the biosynthesis of very long-chain (>C24) saturated fatty acids (VLC-SFA) and very long-chain polyunsaturated fatty acids (VLC-PUFA), had not been characterized in this species. Here, we investigate the repertoire and function of two Elovl4 in tambaqui. Furthermore, we present the first draft genome assembly from tambaqui, and demonstrated the usefulness of this resource in nutritional physiology studies by isolating one of the tambaqui elovl4 genes. Our results showed that, similarly to other teleost species, two elovl4 gene paralogs termed as elovl4a and elovl4b, are present in tambaqui. Tambaqui elovl4a and elovl4b have open reading frames (ORF) of 948 and 912 base pairs, encoding putative proteins of 315 and 303 amino acids, respectively. Functional characterization in yeast showed that both Elovl4 enzymes have activity toward all the PUFA substrates assayed (18:3n-3, 18:2n-6, 18:4n-3, 18:3n-6, 20:5n-3, 20:4n-6, 22:5n-3, 22:4n-6 and 22:6n-3), producing elongated products of up to C36. Moreover, both Elovl4 were able to elongate 22:5n-3 to 24:5n-3, a key elongation step required for the synthesis of docosahexaenoic acid via the Sprecher pathway.
Asunto(s)
Elongasas de Ácidos Grasos/genética , Ácidos Grasos Insaturados/metabolismo , Proteínas de Peces/genética , Peces/metabolismo , Secuencia de Aminoácidos , Animales , Brasil , Clonación Molecular , Ácidos Docosahexaenoicos/biosíntesis , Elongasas de Ácidos Grasos/aislamiento & purificación , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/genética , Proteínas de Peces/metabolismo , Genoma , Sistemas de Lectura Abierta , Filogenia , Alineación de SecuenciaRESUMEN
The Japanese eel Anguilla japonica is a catadromous fish species with considerable farming scale. Previous studies showed that dietary α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) satisfied essential fatty acid requirements in eel, which suggested that Japanese eel should have a complete pathway for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA). However, existing knowledge was insufficient to explain the molecular basis of LC-PUFA biosynthetic capacity in eel. In order to further characterize this pathway in eel, a full-length cDNA of a putative fatty acyl elongase was isolated, with the ORF encoding a protein with 294 amino acids. The putative elongase displayed high homology to Elovl2 of other teleosts. Functional characterization by heterologous expression in yeast showed the protein product of the cDNA had high activity towards C20 and C22 PUFA substrates and low activity towards C18 PUFA substrates, characteristic of Elovl2 elongases. Tissue distribution of the elovl2 mRNA showed highest expression in brain and eyes, which was different from freshwater and anadromous species. This may reflect an important role for this enzyme in the in situ endogenous biosynthesis of docosahexaenoic acid (DHA) in neural tissues in eel. This is the first report of an Elovl2 in a catadromous teleost and demonstrates that Japanese eel has a complete enzyme repertoire required for the endogenous biosynthesis of DHA via the Sprecher pathway. These data have increased our knowledge of the diversity of LC-PUFA biosynthesis in vertebrates, and provided further insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleost fish.
Asunto(s)
Anguilla , Clonación Molecular , Ácidos Docosahexaenoicos , Elongasas de Ácidos Grasos , Proteínas de Peces , Ácido alfa-Linolénico , Anguilla/genética , Anguilla/metabolismo , Animales , Ácidos Docosahexaenoicos/biosíntesis , Ácidos Docosahexaenoicos/genética , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Ácido alfa-Linolénico/genética , Ácido alfa-Linolénico/metabolismoRESUMEN
Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (>C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2.
Asunto(s)
Clonación Molecular , Grasas de la Dieta/farmacología , Elongasas de Ácidos Grasos , Proteínas de Peces , Regulación de la Expresión Génica/efectos de los fármacos , Atún , Animales , Elongasas de Ácidos Grasos/biosíntesis , Elongasas de Ácidos Grasos/genética , Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Atún/genética , Atún/metabolismoRESUMEN
As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the 'Sprecher pathway'. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs.
Asunto(s)
Bivalvos/metabolismo , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Animales , Bivalvos/enzimología , Ácidos Docosahexaenoicos/biosíntesis , Genoma , Moluscos/genética , FilogeniaRESUMEN
Tetracosahexaeoic acid (THA; 24:6n-3) is thought to be the immediate precursor of DHA in rodents; however, the relationship between THA and DHA metabolism has not been assessed in vivo. Here, we infused unesterified 2H5-THA and 13C22-DHA, at a steady state, into two groups of male Long-Evans rats and determined the synthesis-secretion kinetics, including daily synthesis-secretion rates of all 20-24 carbon n-3 PUFAs. We determined that the synthesis-secretion coefficient (a measure of the capacity to synthesize a given fatty acid) for the synthesis of DHA from plasma unesterified THA to be 134-fold higher than for THA from DHA. However, when considering the significantly higher endogenous plasma unesterified DHA pool, the daily synthesis-secretion rates were only 7-fold higher for DHA synthesis from THA (96.3 ± 31.3 nmol/d) compared with that for THA synthesis from DHA (11.4 ± 4.1 nmol/d). Furthermore, plasma unesterified THA was converted to DHA and secreted into the plasma at a 2.5-fold faster rate than remaining as THA itself (26.2 ± 6.3 nmol/d), supporting THA's primary role as a precursor to DHA. In conclusion, using a 3 h infusion model in rats, we demonstrate for the first time in vivo that DHA is both a product and a precursor to THA.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Animales , Semivida , Hidrólisis , Marcaje Isotópico , Cinética , Masculino , RatasRESUMEN
Docosahexaenoic acid (DHA) plays important physiological roles in vertebrates. Studies in rats and rainbow trout confirmed that DHA biosynthesis proceeds through the so-called "Sprecher pathway", a biosynthetic process requiring a Δ6 desaturation of 24:5n-3 to 24:6n-3. Alternatively, some teleosts possess fatty acyl desaturases 2 (Fads2) that enable them to biosynthesis DHA through a more direct route termed the "Δ4 pathway". In order to elucidate the prevalence of both pathways among teleosts, we investigated the Δ6 ability towards C24 substrates of Fads2 from fish with different evolutionary and ecological backgrounds. Subsequently, we retrieved public databases to identify Fads2 containing the YXXN domain responsible for the Δ4 desaturase function, and consequently enabling these species to operate the Δ4 pathway. We demonstrated that, with the exception of Δ4 desaturases, fish Fads2 have the ability to operate as Δ6 desaturases towards C24 PUFA enabling them to synthesise DHA through the Sprecher pathway. Nevertheless, the Δ4 pathway represents an alternative route in some teleosts and we identified the presence of putative Δ4 Fads2 in a further 11 species and confirmed the function as Δ4 desaturases of Fads2 from medaka and Nile tilapia. Our results demonstrated that two alternative pathways for DHA biosynthesis exist in teleosts.
Asunto(s)
Ácidos Docosahexaenoicos/biosíntesis , Peces/metabolismo , Redes y Vías Metabólicas , Animales , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Peces/clasificación , Peces/genética , FilogeniaRESUMEN
Polyunsaturated fatty acids (PUFAs) have been acknowledged as essential nutrients for cephalopods but the specific PUFAs that satisfy the physiological requirements are unknown. To expand our previous investigations on characterisation of desaturases and elongases involved in the biosynthesis of PUFAs and hence determine the dietary PUFA requirements in cephalopods, this study aimed to investigate the roles that a stearoyl-CoA desaturase (Scd) and an elongation of very long-chain fatty acid 4 (Elovl4) protein play in the biosynthesis of essential fatty acids (FAs). Our results confirmed the Octopus vulgaris Scd is a ∆9 desaturase with relatively high affinity towards saturated FAs with ≥ C18 chain lengths. Scd was unable to desaturate 20:1n-15 (∆520:1) suggesting that its role in the biosynthesis of non-methylene interrupted FAs (NMI FAs) is limited to the introduction of the first unsaturation at ∆9 position. Interestingly, the previously characterised ∆5 fatty acyl desaturase was indeed able to convert 20:1n-9 (∆1120:1) to ∆5,1120:2, an NMI FA previously detected in octopus nephridium. Additionally, Elovl4 was able to mediate the production of 24:5n-3 and thus can contribute to docosahexaenoic acid (DHA) biosynthesis through the Sprecher pathway. Moreover, the octopus Elovl4 was confirmed to play a key role in the biosynthesis of very long-chain (>C24) PUFAs.
Asunto(s)
Acetiltransferasas/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Octopodiformes/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas de Peces/metabolismo , Alineación de SecuenciaRESUMEN
The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) provides an intriguing example on how multi-enzymatic cascades evolve. Essential LC-PUFA, such as arachidonic, eicosapentaenoic, and docosahexaenoic acids (DHA), can be acquired from the diet but are also endogenously retailored from C18 precursors through consecutive elongations and desaturations catalyzed, respectively, by fatty acyl elongase and desaturase enzymes. The molecular wiring of this enzymatic pathway defines the ability of a species to biosynthesize LC-PUFA. Exactly when and how in animal evolution a functional LC-PUFA pathway emerged is still elusive. Here we examine key components of the LC-PUFA cascade, the Elovl2/Elovl5 elongases, from amphioxus, an invertebrate chordate, the sea lamprey, a representative of agnathans, and the elephant shark, a basal jawed vertebrate. We show that Elovl2 and Elovl5 emerged from genome duplications in vertebrate ancestry. The single Elovl2/5 from amphioxus efficiently elongates C18 and C20 and, to a marked lesser extent, C22 LC-PUFA. Lamprey is incapable of elongating C22 substrates. The elephant shark Elovl2 showed that the ability to efficiently elongate C22 PUFA and thus to synthesize DHA through the Sprecher pathway, emerged in the jawed vertebrate ancestor. Our findings illustrate how non-integrated "metabolic islands" evolve into fully wired pathways upon duplication and neofunctionalization.
Asunto(s)
Acetiltransferasas/genética , Evolución Molecular , Proteínas de Peces/genética , Lampreas/genética , Anfioxos/genética , Tiburones/genética , Acetiltransferasas/metabolismo , Animales , Proteínas de Peces/metabolismo , Duplicación de Gen , Lampreas/metabolismo , Anfioxos/enzimología , Tiburones/metabolismoRESUMEN
Arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are the most biologically active polyunsaturated fatty acids, but their biosyntheses in mammals are very limited. The biosynthesis of DHA is the most difficult, because this undergoes the Sprecher pathway--a further elongation step from docosapentaenoic acid (DPA), a Δ6-desaturase acting on a C24 fatty acid substrate followed by a peroxisomal chain shortening step. This paper reports the successful heterologous expression of two non-mammalian genes (with modification of codon usage), coding for Euglena gracilis Δ4-desaturase and Siganus canaliculatus Δ4-desaturase respectively, in mammalian cells (HEK293 cell line). Both of the Δ4-desaturases can efficiently function, directly converting DPA into DHA. Moreover, the cooperation of the E. gracilis Δ4-desaturase with C. elegans Δ15-desaturase (able to convert a number of n-6 PUFAs to their corresponding n-3 PUFAs) in transgenic HEK293 cells made a more desirable fatty acid composition--a drastically reduced n-6/n-3 PUFAs ratio and a high level of DHA as well as EPA and ARA. Our findings provide a basis for potential applications of the gene constructs for expression of Δ15/Δ4-desaturases in transgenic livestock to produce such a fatty acid profile in the related products, which certainly will bring benefit to human health.
Asunto(s)
Caenorhabditis elegans/genética , Euglena gracilis/genética , Ácido Graso Desaturasas/genética , Ácidos Grasos Insaturados/metabolismo , Peces/genética , Técnicas de Transferencia de Gen , Animales , Caenorhabditis elegans/enzimología , Cromatografía de Gases , Cartilla de ADN/genética , Euglena gracilis/enzimología , Ácido Graso Desaturasas/metabolismo , Células HEK293 , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally characterized fatty acyl desaturase and elongase cDNAs corresponding to Δ4fad (with some Δ5 activity for the n-3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to higher or lower dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Δ4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Δ4fad was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway.
Asunto(s)
Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Proteínas de Peces/metabolismo , Peces Planos/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Ácido Graso Desaturasas/clasificación , Ácido Graso Desaturasas/genética , Elongasas de Ácidos Grasos , Ácidos Grasos/análisis , Ácidos Grasos Insaturados/análisis , Proteínas de Peces/clasificación , Proteínas de Peces/genética , Peces Planos/genética , Peces Planos/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Lípidos/química , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de TiempoRESUMEN
Most enveloped viruses fuse with host cells and catalyze fusion among host cells by expression of specific patterns of N-glycosylation on their envelope proteins. In the 1970s, it was observed that 2-deoxy-D-glucose (2DOG) and 2-fluoro-2-deoxy-D-mannose (2F2DOM) inhibited N-glycosylation of asparagine (Asn) sites on the external domain of viral envelope proteins. This effect led to the virus particles being non-fusogenic with greatly reduced infectivity and reduced ability to pass from cell to cell by catalyzing cell--cell fusion. At that time, this observation was not particularly important because viral diseases were readily prevented by vaccines and there was no known link between fusogenic viruses and cancer. Today, we are faced with a chronic and lethal viral disease (AIDS) caused by a virus (HIV) that mutates so quickly that we have not been able to produce a vaccine. Moreover, it is spreading among millions of people unable to afford more than basic medications. In addition, cell--cell fusion has been identified as an important, if not essential, step in the progression of abnormal cell clones to clinically significant cancer and fusogenic viruses have been shown to cause progression of some tumors. Here, we reiterate the hypothesize (first made in 1986 by Blough et al. [Blough HA, Pauwels R, De Clercq E, Cogniaux J, Sprecher-Goldberger S, Thiry L. Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins. Biochem Biophys Res Commun 1986; 141:33-8]) that 2DOG, 2F2DOM and related compounds, which interfere with normal N-glycosylation of virus envelope proteins, are attractive candidates for anti-fusogenic drugs that can be used against chronic virus diseases and cancers. This analysis also supports the concept of blocking N-acetylglucosaminyl-transferases with chloroquine or other drugs (proposed by Savarino et al. [Savarino A, Lucia MB, Rastrelli E et al. Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors. J Acquir Immune Defic Syndr 2004; 35:223-32]) as an anti-viral approach. These drugs may have broader utility and lower cost than drugs designed specifically to target the gp41 protein of HIV, which have become popular as viral-entry inhibitors for treatment of HIV/AIDS.
Asunto(s)
Antivirales/farmacología , Desoxiglucosa/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Animales , Asparagina/química , Carbohidratos/química , Fusión Celular , Farmacorresistencia Viral/fisiología , Glucólisis , Glicosilación , Inhibidores de Fusión de VIH/farmacología , Humanos , Modelos Biológicos , Modelos Teóricos , Proteínas del Envoltorio Viral/químicaRESUMEN
Docosahexaenoic acid (DHA) has long been recognized for its beneficial effect in humans, but its biosynthetic pathway has not been clearly established until recently. According to Sprecher, in mammals, DHA is synthesized via a retro-conversion process in peroxisomes-the aerobic delta4 desaturation-independent pathway. Recent identification of a Thraustochytrium delta4 desaturase indicates that delta4 desaturation is indeed involved in DHA synthesis in Thraustochytrium. More interestingly, an alternative pathway for DHA biosynthesis-the anaerobic polyketide synthase pathway was also reported recently to occur in Schizochytrium, another member of the Thraustochytriidae. This mini-review attempts to assess the latest research on these distinct pathways for DHA biosynthesis.
Asunto(s)
Ácidos Docosahexaenoicos/metabolismo , Células Eucariotas/enzimología , Animales , Humanos , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Peroxisomas/metabolismoRESUMEN
In mammalian cells, Sprecher has proposed that the synthesis of long-chain PUFA from the 20-carbon substrates involves two consecutive elongation steps, a delta6-desaturation step followed by retroconversion (Sprecher, H., Biochim. Biophys. Acta 1486, 219-231, 2000). We searched the database using the translated sequence of human elongase ELOVL5, whose encoded enzyme elongates monounsaturated and polyunsaturated FA, as a query to identify the enzyme(s) involved in elongation of very long chain PUFA. The database search led to the isolation of two cDNA clones from human and mouse. These clones displayed deduced amino acid sequences that had 56.4 and 58% identity, respectively, to that of ELOVL5. The open reading frame of the human clone (ELOVL2) encodes a 296-amino acid peptide, whereas the mouse clone (Elovl2) encodes a 292-amino acid peptide. Expression of these open reading frames in baker's yeast, Saccharomyces cerevisiae, demonstrated that the encoded proteins were involved in the elongation of both 20- and 22-carbon long-chain PUFA, as determined by the conversion of 20:4n-6 to 22:4n-6, 22:4n-6 to 24:4n-6, 20:5n-3 to 22:5n-3, and 22:5n-3 to 24:5n-3. The elongation activity of the mouse Elovl2 was further demonstrated in the transformed mouse L cells incubated with long-chain (C20- and C22-carbon) n-6 and n-3 PUFA substrates by the significant increase in the levels of 24:4n-6 and 24:5n-3, respectively. This report demonstrates the isolation and identification of two mammalian genes that encode very long chain PUFA specific elongation enzymes in the Sprecher pathway for DHA synthesis.
Asunto(s)
Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Ácidos Grasos Omega-3/biosíntesis , Ácidos Grasos Insaturados/biosíntesis , Secuencia de Aminoácidos , Animales , Northern Blotting , Clonación Molecular , ADN Complementario/genética , Ácido Graso Desaturasas/metabolismo , Elongasas de Ácidos Grasos , Ácidos Grasos Omega-6 , Cromatografía de Gases y Espectrometría de Masas , Vectores Genéticos/genética , Humanos , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Especificidad de Órganos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The biosynthesis of 4,7,10,13,16-22:5 and 4,7,10,13,16,19-22:6 requires that when 6,9,12,15,18-24:5 and 6,9,12,15,18,21-24:6 are produced in microsomes they must move to peroxisomes for partial beta-oxidation. When the 24-carbon acids were incubated with peroxisomes, 22-carbon acids with their first double bond at position 4 accumulated as did those with their first two double bonds at the 2-trans-4-cis-positions (D. L. Luthria, S. B. Mohammed, and H. Sprecher, J. Biol. Chem. 271, 16020-16025, 1996; and B. S. Mohammed, D. L. Luthria, S. P. Baykousheva, and H. Sprecher, Biochem. J., 326, 425-430, 1997). In the study reported here we analyzed the acyl-CoAs that accumulated when peroxisomes were incubated with 5,8,11,14-20:4 and 6,9,12-18:3, a metabolite that would be produced via one cycle of arachidonate degradation via the pathway requiring both NADPH-dependent 2,4-dienoyl-CoA reductase and delta 3,5, delta 2,4-dienoyl-CoA isomerase. With both substrates the acyl-CoAs of 2-trans-4-10:2, 4-10:1, 2-trans-4,7,10-16:4, and 4,7,10-16:3 accumulated. These results further establish that the reductase catalyzes a control step in the peroxisomal degradation of unsaturated fatty acids. It was not possible to detect any 18- or 12-carbon acyl-CoA when arachidonate was the substrate, nor did any 12-carbon catabolite accumulate from 6,9,12-18:3. The fractional amount of 5,8-14:2 and arachidonate catabolized via the pathway using only the enzymes of saturated fatty acid degradation versus the pathway that also uses the reductase and the isomerase could thus not be estimated.
Asunto(s)
Acilcoenzima A/metabolismo , Ácido Araquidónico/metabolismo , Hígado/metabolismo , Microcuerpos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Ácido gammalinolénico/metabolismo , Animales , Radioisótopos de Carbono , Isomerasas de Doble Vínculo Carbono-Carbono/metabolismo , Ácido Graso Desaturasas/metabolismo , Cinética , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Proteolysis is a key feature of programmed cell death. Extracellular proteinases can activate cell surface receptors which trigger apoptosis, and the effector machinery requires the activation and activity of numerous intracellular proteinases (primarily caspases). Effective control of proteolysis is essential for homeostasis and can occur at two levels: regulation of proteinase activation, and regulation of the activated proteinase. Serpins control activated proteinases and several have been implicated in the regulation of cell death. Serpins that inhibit intracellular processes include the viral proteins CrmA and SPI-1, as well as the granzyme B inhibitor, PI-9. Another endogenous serpin, PN-I, prevents the delivery of an apoptotic signal by inhibiting an extracellular proteinase from cleaving a cell surface receptor. There is evidence to suggest that PAI-2 may target an extracellular as well as an intracellular proteinase. Much of our knowledge of proteolysis within apoptotic cells has come from studies using the poxvirus serpin CrmA/SPI-2. CrmA prevents cytokine processing by inhibiting caspase-1, and protects against Fas-, TNF- and TRAIL-mediated apoptosis by inhibiting an unidentified proteinase specific to these pathways. Work with CrmA has also clearly demonstrated that there are separable effector mechanisms within cells, and that those triggered by growth factor withdrawal, matrix dissociation or cytotoxic ligands are different in several respects to those triggered by radiation, chemicals or steroid hormones. It is likely that analysis of other poxvirus serpins with different inhibitory profiles (especially SPI-1) will yield further insights into these processes. Prospecting for intracellular serpin genes in other virus species may also be fruitful. Finally, all of the serpins known to regulate intracellular proteolysis are members of the ovalbumin subgroup. It remains to be seen whether the more recently described "orphan" ovalbumin serpins (Riewald and Schleef 1995; Sprecher et al. 1995; Sun et al. 1997) also have roles in the regulation of cell death.
Asunto(s)
Apoptosis , Homeostasis , Inhibidores de Serina Proteinasa , Serpinas/fisiología , Animales , Evolución Biológica , Humanos , Serpinas/químicaRESUMEN
It has been proposed that synthesis of docosahexaenoic acid (22:6(n-3) in rat hepatocytes occurs by a route independent of delta 4-desaturase, which involves delta 6-desaturation and retroconversion (Voss A., Reinhart M., Sankarappa S. and Sprecher H. (1991) J. Biol. Chem. 266, 19995-20000). However, most cells exhibit these enzymatic activities and nevertheless synthesize low to undectectable amounts of 22:6(n-3). Moreover, there are few data on the occurrence of this pathway in human cells. In the present work, we have analysed the biosynthetic pathway of 22:6(n-3) in human Y-79 retinoblastoma and Jurkat T-cells. Y-79 cells were supplemented with 18:3(n-3) and 20:5(n-3) or incubated with [1-14C]18:3(n-3) and [1-14C]20:5(n-3) and lipids analysed by argentation TLC, reverse-phase TLC and GLC-mass spectrometry. Pulse-chase experiments revealed that synthesis of 22:6(n-3) from 20:5(n-3) in Y-79 cells occurred through two successive elongations, followed by a delta 6-desaturation of 24:5(n-3) to 24:6(n-3) and retroconversion to 22:6(n-3). Incubation of Y-79 cells with [1-14C]18:3(n-3) in medium containing 50 microM trans-9,12-18:2, a potent inhibitor of delta 6-desaturase, caused a reduction of 22:6(n-3) synthesis mainly by interfering with the desaturation of 18:3(n-3). However, when [1-14C]20:5(n-3) was used as precursor, synthesis of 22:6(n-3) was depressed to a lesser extent and mainly by reduction of 24:6(n-3) retroconversion. Neuronal differentiation of Y-79 cells caused a great increase in delta 6-desaturase activity on 18:3(n-3), though the amount of 22:6(n-3) synthesized did not change or diminish, suggesting the existence of a particular delta 6-desaturase involved in the synthesis of 22:6(n-3). The existence of a distinctive delta 6-desaturase activity could also explain why Jurkat cells growing in serum-free medium showed a near 3-fold increase in the synthesis of pentaenes from 18:3(n-3) and, at the same time, a large decrease in the synthesis of 22:6(n-3). The verification of the involvement of two delta 6-desaturase activities in 22:6(n-3) synthesis would have important implications for the formulation of the nutritional requirements of this fatty acid during development.