1. Introduction
Certain long-chain (C
20–24) polyunsaturated fatty acids (LC-PUFA), namely eicosapentaenoic acid (EPA; 20:5n-3), arachidonic acid (ARA; 20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3), are regarded as physiologically essential for the correct development of vertebrates, including fish [
1]. These compounds can be obtained through the diet or, alternatively, biosynthesized from C
18 polyunsaturated fatty acids (PUFA), such as α-linolenic acid (18:3n-3) and linoleic acid (18:2n-6), via enzymatic reactions carried out by fatty acyl desaturases (Fads) and elongation of very long-chain fatty acid (Elovl) proteins [
1,
2]. Fads are enzymes that introduce double bonds (unsaturations) into PUFA substrates. On the other hand, Elovl are considered to be pivotal components of fatty acid (FA) synthetic pathways [
3,
4], being responsible for a condensation reaction, which results in the extension of the pre-existing FA chain with two new carbon atoms [
1]. The Elovl protein family contains several members [
2,
3,
4], of which only a few have been demonstrated to have PUFA as substrates. Of these, Elovl2, Elovl4, and Elovl5, have well-established roles in the biosynthesis of LC-PUFA in vertebrates [
2,
3,
4], while a novel Elovl8 has been more recently suggested to be also involved in PUFA elongation [
5,
6]. While Elovl2 and Elovl5 are primarily involved in elongation steps within the LC-PUFA biosynthesis pathway, Elovl4 catalyzes the synthesis of very long-chain (>C
24) PUFA (VLC-PUFA), which can have up to 36 or 38 carbons [
1,
7]. Furthermore, Elovl4 is additionally responsible for the production of very long-chain saturated fatty acids (VLC-SFA) [
8].
Virtually all teleosts possess at least two Elovl4 isoforms termed as “Elovl4a” and “Elovl4b” [
2,
9]. Gene expression data indicates that both
elovl4 paralogs have widespread tissue distribution, with
elovl4a being highly expressed in brain [
9,
10] and
elovl4b in eye (retina) and gonads [
9,
10]. The functions of Elovl4a and Elovl4b seem to vary among species. For instance, in zebrafish (
Danio rerio), Elovl4a showed the ability to elongate saturated FA (SFA) to produce VLC-SFA, while only Elovl4b was able to elongate PUFA substrates to produce VLC-PUFA [
9]. However, studies performed on African catfish
(Clarias gariepinus) [
10] and black seabream (
Acanthopagrus schlegelii) [
11] have demonstrated that both Elovl4a and Elovl4b have the ability to biosynthesize VLC-PUFA. These results suggest that the investigation of Elovl4 proteins in teleosts requires a species-specific approach.
The gilthead seabream (
Sparus aurata) and Senegalese sole (
Solea senegalensis) are two commercially important species in marine finfish aquaculture. A recent study highlighed a relationship between the expression of
elovl4 genes in both species and the formation of neural tissues during early life-cycle development [
12]. Indeed, Elovl4 products, i.e., VLC-SFA and VLC-PUFA, play crucial roles during the early-development of vertebrates by guaranteeing the correct development and functionality of the rapidly forming nervous system, where these compounds accumulate [
8,
12]. From what is known in higher vertebrates, VLC-PUFA are generally incorporated into phosphatidylcholine in the photoreceptor cells that make up the retina [
7], and are then bioconverted into elovanoids, which participate in photoreceptor protection [
8,
13]. On the other hand, VLC-SFA are mainly incorporated into sphingolipids in the brain [
10], taking part in the membrane fusion of synaptic vesicles that occur during the neurotransmission process in mammals [
14,
15]. Finally, Elovl4, including teleost Elovl4, can also play a role in the biosynthesis of LC-PUFA, specifically DHA [
16,
17], which is the most abundant FA in brain and retinal cells [
18,
19,
20].
It is crucial to understand the capacity that a given species has for endogenous production of these essential nutrients due to the importance of very long-chain fatty acids (VLC-FA) during early development. Such ability is itself dependent on the complement of
elovl4 genes and the functions of their corresponding encoded enzymes [
1]. Having this in mind, the aim of the present study was to characterize, both molecularly and functionally,
elovl4 paralogs from
S. aurata (
Sa) and
S. senegalensis (
Ss). Previous studies investigating the functions of
fads- and other
elovl-like genes confirmed that both species operate different LC-PUFA biosynthesis mechanisms [
21,
22,
23,
24], especially with regard to the production of DHA. In particular,
Sa operates the so-called “Sprecher pathway” [
21,
25], whereas
Ss produces DHA via the more direct “Δ4 pathway” [
24] (
Figure 1). We will discuss our results in the context of the biosynthetic particularities of both species when considering that both the LC-PUFA and VLC-PUFA biosynthetic pathways are interdependent.
3. Discussion
Sequence analyses revealed that the investigated predicted Elovl4 proteins contain all the characteristic domains of vertebrate Elovl4 family members [
27,
28], including the ER retention signal (RXKXX), the histidine box (HXXHH), which is involved in the electron transfer process during fatty acid elongation [
4], and other transmembrane domains, similarly to what has been described in other fish species [
9,
10,
11,
29,
30,
31,
32,
33,
34,
35]. Furthermore, phylogenetic analysis confirmed the existence of two isoforms of
Sa and
Ss Elovl4, which clustered, together with corresponding Elovl4 orthologs from other teleosts, into different branches, thus confirming that the described Elovl4 isoforms are true orthologs of the Elovl4a and Elovl4b proteins that are present in teleosts [
2]. The conservation of both Elovl4 isoforms in fish genomes [
2,
9,
10,
11,
17,
33] and their clear segregation into separate clusters points towards a likely functional specialization of these proteins in teleosts [
30,
31], which we aimed to further elucidate in this study by functionally characterizing the two isoforms in two new fish species with diverse life histories, dietary habits, and notably different LC-PUFA biosynthesis mechanisms [
21,
22,
23,
24].
Our results support the notion that both isoforms can participate in VLC-SFA elongation, as suggested in previous studies with other fish species including
D. rerio [
9],
C. gariepinus [
10] and Atlantic salmon (
Salmo salar) [
32]. Nevertheless, Elovl4a seems to be more efficient than Elovl4b at elongating VLC-SFA, similarly to what was reported in
D. rerio [
9]. This was particularly evident in
Sa, where the comparison of the SFA profile of yeast transformed with
elovl4a or
elovl4b with that of control yeast revealed that Elovl4b was only active in the elongation step from 26:0 to 28:0, whereas Elovl4a was clearly able to significantly elongate SFA substrates from 26:0 up to 32:0. The functional characterization of
Ss Elovl4 enzymes showed some differences with respect to
Sa, particularly concerning the preferred fatty acid substrates. Whereas the
Sa Elovl4 isoforms have 26:0 as the most preferred precursor for VLC-SFA biosynthesis, <C
24 saturated fatty acids appear to be more adequate for the
Ss Elovl4 isoforms. This could be related to differences in VLC-SFA requirements between the two fish species [
12], but further studies are necessary in order to clearly establish this.
Functional analyses of Elovl4a and Elovl4b confirmed that both proteins actively participate in the biosynthesis of either n-6 or n-3 VLC-PUFA, from n-6 and n-3 PUFA substrates, in the two studied fish species. These results are in agreement with Elovl4 functional characterization studies in other fish species, which showed similar elongation capabilities [
9,
10,
11,
29,
32,
34,
35], consistent with the functionality that is described in mammals [
36] and other aquatic organisms, such as common octopus (
Octopus vulgaris) [
37]. However, intra- and inter-specific differences were found in the efficiency of the different Elovl4 isoforms to biosynthesize VLC-PUFA. In this respect,
Sa Elovl4a showed a clearly higher affinity towards the elongation of n-6 PUFA substrates, while
Sa Elovl4b was particularly active towards n-3 PUFA substrates. On the other hand, only Elovl4b was able to elongate DHA, up to 34:6n-3. Similar results were obtained in Senegalese sole, where both of the isoforms were active towards n-6 or n-3 PUFA substrates, in this case with less clear differences in terms of substrate preference, but only Elovl4b showed activity towards DHA.
Interestingly, although fish Elov4b seem to have a predominant role in the DHA biosynthesis pathway [
10,
11,
29], both
Sa Elovl4 isoforms had the ability to elongate 20:5n-3 and 22:5n-3 to 24:5n-3, which is a key intermediate FA in the biosynthesis of DHA via the ∆6 “Sprecher pathway” [
25] occurring in this species [
21,
22]. Similarly, both
Ss Elovl4 isoforms, although Elovl4b more prominently, had the capacity to produce 22:5n-3 from 20:5n-3. This is a key substrate for DHA biosynthesis via the ∆4-desaturation pathway that was carried out by
Ss Fads2 [
24]. This redundancy in an activity that is central for DHA biosynthesis [
9,
11,
17,
30,
33,
35] highlights the essentiality of this compound for the correct development and survival of marine fish [
1]. It is well known that the correct biosynthesis of DHA is crucial for the normal development of the fish cognitive system, especially during early stages, when its deficiency can cause visual and/or neural damage [
18,
38]. Moreover, it is highly conceivable that the conservation of two Elovl4 enzymes with the ability to elongate 22:5n-3 and 24:5n-3 can confer a substantial adaptive advantage in marine fish species that have lost the
elovl2 gene during evolution [
1,
2,
11].
It is also noteworthy that, similar to what has been described in zebrafish [
9],
Sa and
Ss Elovl4a elongases both showed low elongation activity from DHA to 24:6n-3. This could suggest that, as described in rat retinas [
39], EPA and not DHA might be the preferred substrate for VLC-PUFA biosynthesis in fish. This assumes the formation of VLC-PUFA hexaenes from LC-PUFA pentaenes via Δ6 desaturation of 24:5n-3, which should only take place in the case of gilthead seabream, since Senegalese sole is believed to lack this desaturation capacity in favour of a Δ4 Sprecher-independent pathway for DHA biosynthesis [
24]. Paradoxically, our functional results revealed a higher capacity for 24:5n-3 production in this latter species. On the other hand, similarly to what has been found in other teleosts [
10,
11,
29], Elovl4b proteins in both species were able to elongate 24:6n-3 up to 32:6n-3, a VLC-PUFA found in retinal phosphatidylcholine in fish [
40,
41]. Thus, this specific activity of Elovl4b proteins, along with the above-mentioned presence of 32:6n-3 in fish retina, is coherent with the tissue expression results obtained for both species, in which Elovl4b transcripts were mostly found in the eye suggesting that, similarly to what has been described in other teleosts, like
T. thynnus [
29],
D. rerio [
9],
A. schlegelli [
11], rainbow trout (
Oncorhynchus mykiss) [
31],
S. salar [
32], or orange-spotted grouper (
Epinephelus coioides) [
35]; this is a major tissue for VLC-PUFA biosynthesis.
The quantitative expression results confirmed previous evidences of a differential
elovl4a and
elovl4b tissue-specific expression pattern [
12,
42], with
elovl4a being mostly expressed in fish brain [
9,
10,
11,
31], and
elovl4b in eye [
9,
11,
29,
31,
32,
35]. Therefore, in spite of the specific functions of VLC-PUFA still not being fully understood in vertebrates [
7], and their identification being very scarce in fish [
40,
41], the results reported here in terms of
elovl4 tissue expression in these two fish species suggests a role of these enzymes in the local biosynthesis and the incorporation of VLC-FA in fish neural tissues. This is in agreement with what is known in mammals [
8], in which VLC-PUFA are key functional components, essential for the development and cell protection, of neural tissues such as those found in retina or brain [
7,
13,
36]. More specifically, certain VLC-PUFA are synthesized and esterified at the
sn-1 position of the glycerol backbone of phosphatidylcoline, which is then deposited in retinal photoreceptors, where it plays an important neuroprotective role [
7,
8,
43]. Other VLC-SFA are mainly incorporated into sphingolipids in the central nervous system [
8], playing a key role in the membrane fusion of synaptic vesicles occurring during neurotransmission processes [
14,
15].
The application of this knowledge is of special relevance during early larval development, particularly in species with high commercial interest for aquaculture production, as is the case of gilthead seabream and Senegalese sole, and it should be kept in mind in feeding protocols during hatchery rearing. Mammalian
elovl4 expression is developmentally regulated in the brain, with expression peaking around the time of birth and falling as the brain matures [
44], thus pointing to a prominent role of this protein in neurogenesis [
8]. In fish species, it is equally likely that the optimal functioning of Elovl4 enzymes is particularly critical during early developmental stages, at a time when neural tissues are rapidly forming [
20,
45], in order to ensure correct biosynthesis and tissue accumulation of VLC-PUFA products [
9,
12,
30,
42]. Hence, not surprisingly,
elovl4 genes were found to be widely expressed in neural tissues (brain mass and eyes) during the embryonic phase of
D. rerio [
9] and cobia (
Rachycentron canadum) [
30]. Moreover, as previously described [
12], retinogenesis in gilthead seabream and Senegalese sole larvae is clearly synchronized with an increase in expression of both
elovl4 genes. Consequently, as described in mammals [
46], alterations in VLC-PUFA biosynthesis could negatively impact visual acuity and disrupt brain functionality, jeopardizing the normal development of fish. Although neural and visual structures of newly hatched fish larvae are undeveloped, cones and rod cells differentiate quite early [
47,
48], and their correct development and functionality is determining for fish larvae to begin feeding exogenously [
49] and, hence, for their survivability. This is particularly relevant in visual predators, such as gilthead seabream and Senegalese sole, which previously showed a differential
elovl4 expression in larvae and postlarvae according to the VLC-PUFA putative needs associated with each life-stage and LC-PUFA dietary availability [
12,
42]. Finally, the results from the present study evidencing a low activity of Elovl4 on DHA and a higher activity on longer (26 and 28 C) substrates, which are, in turn, dependent on DHA, reinforce the idea that an appropriately high dietary supply of DHA is critical in early stages of fish larval life, not only “per se”, i.e., related to the essential nature of this fatty acid on its own, but also as a bottleneck substrate for subsequent VLC-PUFA synthesis.
In view of the results that are presented here, we conclude that both gilthead seabream and Senegalese sole possess two distinct Elovl4-like elongases named Elovl4a and Elovl4b based on their homology to the zebrafish orthologs [
9]. Functional analyses denoted that, although with some specificities, both
Sa and
Ss Elovl4a and Elovl4b are involved in VLC-SFA and VLC-PUFA biosynthesis, being able to elongate a range of substrates up to C
34 VLC-SFA and VLC-PUFA. Moreover, neural tissues are the major site of
elovl4 expression, with brain and eye exhibiting the highest
elovl4a and
elovl4b expression levels, respectively. Therefore, these are likely the main tissues of VLC-FA biosynthesis and accumulation, which highlights the importance of these compounds for crucial physiological processes, such as vision and brain function, particularly during early fish development.