In silico proteolysis and analysis of bioactive peptides from sequences of fatty acid desaturase 3 (FAD3) of flaxseed protein.

Flaxseed (Linum usitatissimum), commonly known as linseed is an oilseed crop, emerging as an important and functional ingredient of food and has been paid more attention due to its nutritional value as well as beneficial effects. It is mainly rich in is α-linolenic acid (ALA, omega-3 fatty acid), fibres and lignans that have potential health benefits in reducing cardiovascular diseases, diabetes, osteoporosis, atherosclerosis, cancer, arthritis, neurological and autoimmune disorders. Due to its richness in omega-3 fatty acid, a group of enzymes known as fatty acid desaturases (FADs) mainly introduce double bonds into fatty acids' (FAs) hydrocarbon chains that produce unsaturated fatty acids. Fatty acid desaturase 3 (FAD3), the commonest microsomal enzyme of omega-3 fatty acid, synthesizes linolenic acid (C18:3) from linoleic acid located in endoplasmic reticulum (ER) facing towards the cytosol. The emerging field of bioinformatics and large number of databases of bioactive peptides, helps in providing time-saving and efficient method for identification of potential bioactivities of any protein. In this study, 10 unique sequences of FAD3 from flaxseed protein have been used for in silico proteolysis and releasing of various bioactive peptides using three plant proteases, namely ficin, papain and stem bromelain, that are evaluated with the help of BIOPEP database. Overall, 20 biological activities were identified from these proteins. The results showed that FAD3 protein is a potential source of peptides with angiotensin-I-converting enzyme (ACE) inhibitory and dipeptidyl peptidase-IV (DPP-IV) activities, and also various parameters such as ∑A, ∑B, AE, W, BE, V and DHt were also calculated. Furthermore, PeptideRanker have been used for screening of novel promising bioactive peptides. Various bioinformatics tools also used to study protein's physicochemical properties, peptide's score, toxicity, allergenicity aggregation, water solubility, and drug likeliness. The present work suggests that flaxseed protein can be a good source of bioactive peptides for the synthesis of good quality and quantity of oil, and in silico method helps in investigating and production of functional peptides.

Identification and functional characterization of Buglossoides arvensis microsomal fatty acid desaturation pathway genes involved in polyunsaturated fatty acid synthesis in seeds.

Buglossoides arvensis seed oil is the richest natural source of stearidonic acid (SDA), an ω-3 fatty acid with nutraceutical potential superior to α-linolenic acid (ALA). The molecular basis of polyunsaturated fatty acid synthesis in B. arvensis is unknown. Here, we describe the identification of B. arvensis fatty acid desaturase2 (BaFAD2), fatty acid desaturase3 (BaFAD3), and Delta-6-desaturase (BaD6D-1 and BaD6D-2) genes by mining the transcriptome of developing seeds and their functional characterization by heterologous expression in Saccharomyces cerevisiae. In silico analysis of their encoded protein sequences showed conserved histidine-boxes and signature motifs essential for desaturase activity. Expression profiling of these genes showed higher transcript abundance in reproductive tissues than in vegetative tissues, and their expression varied with temperature stress treatments. Yeast expressing BaFAD2 was found to desaturate both oleic acid and palmitoleic acid into linoleic acid (LA) and hexadecadienoic acid, respectively. Fatty acid supplementation studies in yeast expressing BaFAD3 and BaD6D-1 genes revealed that the encoded enzyme activities of BaFAD3 efficiently converted LA to ALA, and BaD6D-1 converted LA to γ-linolenic acid and ALA to SDA, but with an apparent preference to LA. BaD6D-2 did not show the encoded enzyme activity and is not a functional D6D. Our results provide an insight into SDA biosynthesis in B. arvensis and expand the repository of fatty acid desaturase targets available for biotechnological production of SDA in traditional oilseed crops.

FADS3 is a Δ14Z sphingoid base desaturase that contributes to gender differences in the human plasma sphingolipidome.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = -log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

Enhancing microRNA167A expression in seed decreases the α-linolenic acid content and increases seed size in Camelina sativa.

Despite well established roles of microRNAs in plant development, few aspects have been addressed to understand their effects in seeds especially on lipid metabolism. In this study, we showed that overexpressing microRNA167A (miR167OE) in camelina (Camelina sativa) under a seed-specific promoter changed fatty acid composition and increased seed size. Specifically, the miR167OE seeds had a lower α-linolenic acid with a concomitantly higher linoleic acid content than the wild-type. This decreased level of fatty acid desaturation corresponded to a decreased transcriptional expression of the camelina fatty acid desaturase3 (CsFAD3) in developing seeds. MiR167 targeted the transcription factor auxin response factor (CsARF8) in camelina, as had been reported previously in Arabidopsis. Chromatin immunoprecipitation experiments combined with transcriptome analysis indicated that CsARF8 bound to promoters of camelina bZIP67 and ABI3 genes. These transcription factors directly or through the ABI3-bZIP12 pathway regulate CsFAD3 expression and affect α-linolenic acid accumulation. In addition, to decipher the miR167A-CsARF8 mediated transcriptional cascade for CsFAD3 suppression, transcriptome analysis was conducted to implicate mechanisms that regulate seed size in camelina. Expression levels of many genes were altered in miR167OE, including orthologs that have previously been identified to affect seed size in other plants. Most notably, genes for seed coat development such as suberin and lignin biosynthesis were down-regulated. This study provides valuable insights into the regulatory mechanism of fatty acid metabolism and seed size determination, and suggests possible approaches to improve these important traits in camelina.

Fatty Acid Desaturase 3 (FADS3) Is a Specific ∆13-Desaturase of Ruminant trans-Vaccenic Acid.

In mammalian species, the Fatty Acid Desaturase (FADS) gene cluster includes FADS1 (∆5-desaturase), FADS2 (∆6-desaturase), and a third gene member, named FADS3. According to its high degree of nucleotide sequence homology with both FADS1and FADS2, FADS3 was promptly suspected by researchers in the field to code for a new mammalian membrane-bound fatty acid desaturase. However, no catalytic activity was attributed to the FADS3 protein for a decade, until the rat FADS3 protein was shown in vitro to be able to catalyze the unexpected ∆13-desaturation of trans-vaccenic acid, producing the trans11,cis13-conjugated linoleic acid isomer. This review summarizes the recent investigations establishing the FADS3 enzyme as a reliable mammalian trans-vaccenate ∆13-desaturase in vivo and tries to identify further unresolved issues that need to be addressed.

Conversion of dietary trans-vaccenic acid to trans11,cis13-conjugated linoleic acid in the rat lactating mammary gland by Fatty Acid Desaturase 3-catalyzed methyl-end Δ13-desaturation.

In vitro, the rat Fatty Acid Desaturase 3 (FADS3) gene was shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of trans-vaccenic acid, producing the trans11,cis13-conjugated linoleic acid (CLA) isomer. FADS3 may therefore be the first methyl-end trans-vaccenate Δ13-desaturase functionally characterized in mammals, but the proof of this concept is so far lacking in vivo. The present study therefore aimed at investigating further the putative in vivo synthesis of trans11,cis13-CLA from dietary trans-vaccenic acid in rodents. During one week of pregnancy and two weeks post-partum, Sprague-Dawley female rats were fed two diets either high (10.0% of fatty acids and 3.8% of energy intake) or low (0.4% of fatty acids and 0.2% of energy intake) in trans-vaccenic acid. The trans11,cis13-CLA was specifically detected, formally identified and reproducibly quantified (0.06% of total fatty acids) in the mammary gland phospholipids of lactating female rats fed the high trans-vaccenic acid-enriched diet. This result was consistent with FADS3 mRNA expression being significantly higher in the lactating mammary gland than in the liver. Although the apparent metabolic conversion is low, this physiological evidence demonstrates the existence of this new pathway described in the lactating mammary gland and establishes the FADS3 enzyme as a reliable mammalian trans-vaccenate Δ13-desaturase in vivo.

Fads3 modulates docosahexaenoic acid in liver and brain.

Fatty acid desaturase 3 (FADS3) is the third member of the FADS gene cluster. FADS1 and FADS2 code for enzymes required for highly unsaturated fatty acid (HUFA) biosynthesis, but FADS3 function remains elusive. We generated the first Fads3 knockout (KO) mouse with an aim to characterize its metabolic phenotype and clues to in vivo function. All mice (wild type (WT) and KO) were fed facility rodent chow devoid of HUFA. No differences in overt phenotypes (survival, fertility, growth rate) were observed. Docosahexaenoic acid (DHA, 22:6n-3) levels in the brain of postnatal day 1 (P1) KO mice were lower than the WT (P < 0.05). The ratio of docosapentaenoic acid (DPA, 22:5n-3) to DHA in P1 KO liver was higher than in WT suggesting lower desaturase activity. Concomitantly, 20:4n-6 was lower but its elongation product 22:4n-6 was greater in the liver of P1 KO mice. P1 KO liver Fads1 and Fads2 mRNA levels were significantly downregulated whereas expression levels of elongation of very long chain 2 (Elovl2) and Elovl5 genes were upregulated compared to age-matched WT. No Δ13-desaturation of vaccenic acid was observed in liver or heart in WT mice expressing FADS3 as was reported in vitro. Taken together, the fatty acid compositional results suggest that Fads3 enhances liver-mediated 22:6n-3 synthesis to support brain 22:6n-3 accretion before and during the brain growth spurt.

Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid.

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11,cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11,cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore co-exist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11,cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11,cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11,cis-13 CLA and cis-9,trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11,cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue.

Histone acetyltransferase general control non-repressed protein 5 (GCN5) affects the fatty acid composition of Arabidopsis thaliana seeds by acetylating fatty acid desaturase3 (FAD3).

Seed oils are important natural resources used in the processing and preparation of food. Histone modifications represent key epigenetic mechanisms that regulate gene expression, plant growth and development. However, histone modification events during fatty acid (FA) biosynthesis are not well understood. Here, we demonstrate that a mutation of the histone acetyltransferase GCN5 can decrease the ratio of α-linolenic acid (ALA) to linoleic acid (LA) in seed oil. Using RNA-Seq and ChIP assays, we identified FAD3, LACS2, LPP3 and PLAIIIß as the targets of GCN5. Notably, the GCN5-dependent H3K9/14 acetylation of FAD3 determined the expression levels of FAD3 in Arabidopsis thaliana seeds, and the ratio of ALA/LA in the gcn5 mutant was rescued to the wild-type levels through the overexpression of FAD3. The results of this study indicated that GCN5 modulated FA biosynthesis by affecting the acetylation levels of FAD3. We provide evidence that histone acetylation is involved in FA biosynthesis in Arabidopsis seeds and might contribute to the optimization of the nutritional structure of edible oils through epigenetic engineering.