Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of four fatty acyl elongases and a novel desaturase capacity

https://doi.org/10.1016/j.bbalip.2019.04.004Get rights and content

Highlights

  • The S. constricta Elovl involved in LC-PUFA synthesis were investigated.

  • The desaturation ability of S. constricta Δ6 Fad toward 24:5n-3 was determined.

  • S. constricta is the first mollusc with capacity to operate the Sprecher pathway.

  • DHA biosynthetic ability was limited to certain marine molluscs.

Abstract

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.

Introduction

In vertebrates, long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially arachidonic acid (20:4n-6, ARA), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), are key components of cellular membranes, determining their properties, and also serve as precursors of eicosanoids, affecting signal transductions [1,2]. Moreover, LC-PUFA and their metabolites are essential for various physiological processes including neurological development and the immune response, and can be beneficial in mitigating several pathologies [[3], [4], [5]]. Provision of LC-PUFA in vertebrates can be via the diet directly or through endogenous production (biosynthesis) from dietary essential C18 polyunsaturated fatty acids (PUFA), linoleic acid (18:2n-6, LA) and α-linolenic acid (18:3n-3, ALA) [6]. Two types of enzymes, namely elongation of very long-chain fatty acid (Elovl) proteins and front-end desaturases play major roles in vertebrate LC-PUFA biosynthetic pathways [6]. Elovl enzymes are rate-limiting enzymes in fatty acid (FA) elongation pathways and catalyze the condensation reaction [6,7]. Of the seven members of the Elovl family (Elovl1–7) described in vertebrates, only Elovl2, Elovl4 and Elovl5 have demonstrated elongation ability toward PUFA substrates [6,7]. Typically, Elovl5 exhibits high activity toward C16–20 PUFA substrates, Elovl2 shows high activity toward C20–22 PUFA, whereas Elovl4 is involved in the biosynthesis of very long-chain PUFA (VLC-PUFA; ˃C24) present especially in retina [8,9]. In addition to elongases, vertebrates possess two distinct front-end desaturases termed Fads1 and Fads2 primarily with Δ5 and Δ6 desaturase activity, respectively [6]. Typically, Fads1 plays a key role in the biosynthesis of ARA and EPA by desaturation of 20:3n-6 and 20:4n-3, respectively. Fads2 is involved in the desaturation of LA and ALA to give 18:3n-6 and 18:4n-3, respectively. Importantly, Fads2 also catalyze the rate-limiting step in DHA biosynthesis by conversion of 24:5n-3 to produce 24:6n-3, which is then partially β-oxidized to DHA via the Sprecher pathway [10].

Unlike vertebrates, the pathways of LC-PUFA biosynthesis in invertebrates have been less investigated [11,12]. However, marine molluscs are arguably the group of aquatic invertebrates in which the biosynthesis of LC-PUFA has been most extensively investigated [[11], [12], [13]], partly driven by their critical roles in aquatic ecology and trophic cascade, as well as their importance as sources of health-promoting LC-PUFA for human consumers [14]. Marine molluscs have been demonstrated to possess genes encoding enzymes with roles in the biosynthesis of LC-PUFA [[11], [12], [13],15]. Interestingly, the biosynthetic capability of LC-PUFA in marine molluscs varies greatly among species and depends highly on their complement of desaturase and elongase genes, as well as their enzymatic activities [12]. Two different types of Elovl with roles in LC-PUFA biosynthesis have been characterized from marine molluscs. More specifically, an enzyme called “Elovl2/5”, regarded as an ancestral protein of the vertebrate Elovl2 and Elovl5 [16], has been characterized from the cephalopods Octopus vulgaris and Sepia officinalis [17,18], and the bivalves Chlamys nobilis and Crassostrea angulate [19,20]. Furthermore, Elovl4 orthologs have been characterized from C. nobilis and O. vulgaris [21,22]. Meanwhile, two different fatty acyl desaturases (Fad) involved in LC-PUFA biosynthesis have been identified from marine molluscs, including Δ5 Fad from O. vulgaris [23], S. officinalis [18], C. nobilis [24] and the gastropod Haliotis discus hannai [25], as well as a Δ8 Fad from C. nobilis [21].

As an economically important bivalve species, the razor clam Sinonovacula constricta, is widely distributed in estuarine and intertidal zones along the coasts of the West Pacific Ocean. It is one of the five principal marine molluscs in the global aquaculture industry, with a total production of over 823,000 tons, with a value of US$ 1.3 billion in 2016 [26]. From a nutritional point of view, S. constricta is a good source of health-promoting omega-3 (or n-3) LC-PUFA, especially DHA, which accounts for about 10% of the total FA [27]. Recently, three Fad were successfully characterized from S. constricta [28], two of which showed Δ5 desaturase activity and will be referred to as “Δ5 Fad_a” and “Δ5 Fad_b”. A third Fad enzyme from S. constricta had Δ6 desaturase activity and will be termed herein as “Δ6 Fad”. Importantly, S. constricta Δ6 Fad, the first report of a Δ6 desaturase in a marine mollusc, was able to desaturate both 18:3n-3 and 18:2n-6, but activity toward 24:5n-3, necessary for the Sprecher pathway [10], was not tested [28]. Moreover, no functional characterization of Elovl was reported in S. constricta.

The present study aimed to systematically characterize the complete repertoire of Elovl with putative roles in LC-PUFA biosynthesis pathway in S. constricta. Here we report on the molecular and functional characterization of four distinct Elovl genes, including one Elovl2/5, two Elovl4 (a and b isoforms) and a novel Elovl (termed Elovl_c). Furthermore, we investigated the ability of S. constricta Δ6 Fad to desaturate 24:5n-3 to 24:6n-3, so that we could establish whether this species has the potential to operate the Sprecher pathway.

Section snippets

Full-length cloning of S. constricta Elovl cDNAs

Total RNA was extracted from mixed tissues including muscle, gill and gonad of adult S. constricta specimens (55.23 ± 3.31 mm × 17.82 ± 1.21 mm, shell length × shell width; mean ± SD, n = 6) using MiniBEST Universal RNA Extraction Kit (TaKaRa, Japan). RNA quality was determined by running a subsample (~600 ng) on a 1% agarose gel and RNA concentration was measured using a NanoDrop® ND-1000 (NanoDrop, USA). One μg of total RNA was reverse transcribed to cDNA using PrimeScript™ RT-PCR Kit

Sequences and phylogenetics of the S. constricta Elovl

The ORF of S. constricta Elovl2/5 was 933 bp encoding a polypeptide of 310 aa (Fig. 1). The ORFs of S. constricta Elovl4_a and Elovl4_b were both 876 bp encoding polypeptides of 291 aa, whereas the ORF of S. constricta Elovl_c contained 912 bp encoding 303 aa (Fig. 1). Notably, S. constricta Elovl4_a and Elovl4_b shared high identity (89.69%) in terms of aa sequence, with the main different aa region between them being highlighted with a bold line square in Fig. 1. The detailed sequences of S.

Discussion

The deduced aa sequences of S. constricta Elovl all contained a diagnostic histidine box, which is conserved in all Elovl family members [30], indicating that Elovls have functional regions highly conserved during evolution. Notably, all four sequences contained a diagnostic “Q” (glutamine) in position −5 from the H**HH, characteristic of PUFA elongases and which is not present in non-PUFA elongases [30], indicating that they all may play possible roles in PUFA elongation. Consistent with this,

Conflict of interest

The authors have no conflict of interest to declare.

Transparency document

Transparency document.

Acknowledgement

This research was supported by the National Key Research and Development Program of China (2018YFD0900702), Ningbo Science and Technology Research Projects, China (2017C110003), Zhejiang Major Science Project, China (2018C02G2201013), the Earmarked Fund for Modern Agro-industry Technology Research System, China (CARS-49), and was partly sponsored by K. C. Wong Magna Fund in Ningbo University. In addition, Zhaoshou Ran especially wants to thank Fei Kong for her patience, care and support in life

References (40)

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