Molecular basis for differential elongation of omega-3 docosapentaenoic acid by. Rheumatology Unit, Royal Adelaide Hospital, SA, Australia

Transcription

1 Moleular asis for differential elongation of omega-3 doosapentaenoi aid y the rat Elovl5 and Elovl Melissa K. Gregory 1, Leslie G. Cleland and Mihael J. James Rheumatology Unit, Royal Adelaide Hospital, SA, Australia 1 To whom orrespondene should e addressed. Melissa Gregory Rheumatology Unit, Royal Adelaide Hospital North Terrae, Adelaide, South Australia, Australia 5000 Tel: Fax: melissa.gregory@health.sa.gov.au Downloaded from y guest, on Otoer 7, 018 Areviated title: Moleular asis for differential elongation ativity Areviations: ALA, α-linoleni aid;, doosapentaenoi aid; DHA, doosahexaenoi aid; EPA, eiosapentaenoi aid; SDA, stearidoni aid; SDM, site direted mutagenesis; TMS, transmemrane segment 1

2 ABSTRACT Funtional haraterisation of the rat elongases, Elovl5 and Elovl, have identified that Elovl is ruial for omega-3 doosahexaenoi aid (DHA; :n-3) synthesis. Whilst the sustrate speifiities of the rat elongases had some overlap, only Elovl an onvert the C omega-3 PUFA doosapentaenoi aid (; :5n-3) to :5n-3 whih is the penultimate preursor of DHA. In order to etter understand the potential for these elongases to e involved in DHA synthesis, we have examined the moleular reasons for the differenes etween Elovl5 and Elovl in their aility to elongate to :5n-3. We identified a region of heterogeneity etween Elovl5 and Elovl spanning transmemrane segments and 7. Using a yeast expression system we examined a series of Elovl/Elovl5 himeras and point mutations to identify Elovl residues within this region whih are responsile for sustrate speifiity. The results indiate that the ysteine at position 17 in Elovl and a tryptophan at the equivalent position in Elovl5 explain their differing ailities to elongate to :5n-3. Further studies onfirmed that Elovl C17 is a ritial residue for elongation of at the level oserved in the native protein. Understanding the Downloaded from y guest, on Otoer 7, 018 aility of elongases to synthesise :5n-3 may provide a asis for using sequene data to predit their aility to ultimately support DHA synthesis. Supplementary key words: himera, desaturase, doosahexaenoi aid, eiosapentaenoi aid, elongase

3 INTRODUCTION Synthesis of the omega-3 C PUFA doosahexaenoi aid (DHA; :n-3) from the C 18 PUFA α-linoleni aid (ALA; 18:3n-3) requires a series of desaturation and elongation reations. Although there is evidene that Δdesaturase is rate-limiting for onversion of ALA to the C 0 PUFA eiosapentaenoi aid (EPA; 0:5n-3), it is not rate-limiting for overall DHA synthesis eause the downstream produts of Δdesaturase, stearidoni aid (SDA; 18:n-3) and EPA, are poorly onverted to DHA (1-). Therefore, we examined the two elongases, Elovl5 and Elovl, whih have een overlooked as regulators of DHA synthesis. Using a yeast expression system, it was apparent that the sustrate speifiities of the two rat elongases had some overlap, ut that only Elovl ould onvert endogenously formed C PUFA doosapentaenoi aid (; :5n-3) to :5n-3 whih is the penultimate preursor of DHA (5). Elovl performs the sequential elongation of EPA to followed y further elongation to :5n-3. Thus, Elovl is ruial for DHA synthesis at least in the rat where Elovl5 annot Downloaded from y guest, on Otoer 7, 018 elongate to :5n-3 (5). This proaly explains the poor or asent aility to produe DHA in speies that do not have detetale Elovl suh as arramundi (, 7) or in speies suh as the rat in whih Elovl is expressed at low levels (5). However, there is not an asolute Elovl dependene for DHA synthesis in all speies eause the sea ream, oia, Atlanti luefin tuna and hiken Elovl5 have a small ut measurale aility to elongate (8-11). In order to etter understand the potential for these elongases to e involved in DHA synthesis, we have sought the moleular 3

4 reasons for the differenes etween Elovl5 and Elovl in their aility to elongate to :5n-3. Purifiation of memrane-ound elongases to determine the sustrate inding poket has proven to e unsuessful (1). However, himeri elongase proteins from yeast (13), the moss Physomitrella patens (1), and the fungi Pythium irregulare and Phytophthore infestans (15) have een used to investigate the regions involved in C 18 and C 0 PUFA sustrate speifiity and produt hain length determination. Therefore, we have onstruted a series of rat Elovl/Elovl5 himeras and point mutations to examine the Elovl residues responsile for sustrate speifiity using a yeast expression system. Downloaded from y guest, on Otoer 7, 018

5 EXPERIMENTAL PROCEDURES Constrution of the himeri elongase protein Amplifiation of the 00 p rat 5'-Elovl (position 1-00 p), 171 p rat 3'-Elovl (position p) and 9 p (position p) rat Elovl5 fragments were performed using the himera 1 primers in supplementary tale I, template pyes- Elovl or pyes-elovl5, respetively (5), and Finnzymes Phusion Hot Start High- Fidelity DNA Polymerase (New England BioLas In., Arundel, Qld, Australia). Cyling onditions were as follows: initial denaturation step at 98 C for 30 s, followed y 5 yles of denaturation at 98 C for 10 s, annealing at 7 C/70 C (Elovl/Elovl5) for 0 s, and extension at 7 C for 15 s, followed y a final extension at 7 C for 5 min. The Elovl PCR produts were gel purified and DpnI digested to remove template, while the 9 p Elovl5 PCR produt was used diretly for susequent amplifiations. Chimera 1 was formed using two steps. Amplifiation of the 5'-Elovl+Elovl5 and Elovl5+3'-Elovl fragments was initially performed with template 5'-Elovl and Elovl5 or Elovl5 and 3'-Elovl, respetively, using the Elovl yling onditions aove. Chimera 1 was then amplified using the Elovl primers Downloaded from y guest, on Otoer 7, 018 ontaining restrition enzyme sites flanking the open reading frame (Supplementary Tale I, himera 1) and template 5'-Elovl+Elovl5 and Elovl5+3'-Elovl. Cyling onditions were as follows: initial denaturation step at 98 C for 30 s, followed y 10 yles of denaturation at 98 C for 10 s, annealing at 5 C-10 C (with eah yle the temperature was redued y 1 C) for 0 s, and extension at 7 C for 15 s, followed y 0 yles of denaturation at 98 C for 10 s, annealing at 5 C for 0 s, and extension at 7 C for 15 s, followed y a final extension at 7 C for 5 min. The himera 1 DNA and the expression vetor pyes (Invitrogen Australia Pty. Ltd., 5

6 Mount Waverley, Vi, Australia) were restrition enzyme treated and ligated using T DNA ligase (1.5 Weiss units) (Promega, WI, USA). Transformation of the resulting onstrut, pyes-himera1 into MAX Effiieny DH5a Competent E. oli ells (Invitrogen Australia Pty. Ltd.) was performed using heat-shok. Putative transformants were seleted using 100 mg ml -1 ampiillin and PCR sreening. Reominant plasmids were purified and sequened at the Institute of Medial and Veterinary Siene (Adelaide, Australia). Site direted mutagenesis of the himeri elongase, Elovl5 or Elovl Site direted mutagenesis (SDM) was used to hange Elovl5 amino aids in himeri protein 1 ak to the equivalent Elovl amino aids. A series of SDM resulted in the onstrution of himeri proteins -5. Individual amino aid hanges were also made in Elovl5 or Elovl using SDM. Complementary primers with a minimum of twelve ase pairs either side of the introdued mutation were designed (supplementary tale I). PCR amplifiation was performed using the primers and orresponding template outlined in supplementary tale I and Finnzymes Phusion Hot Start High-Fidelity DNA Polymerase (New England BioLas In.). Cyling onditions were as follows: Downloaded from y guest, on Otoer 7, 018 initial denaturation step at 98 C for 30 s, followed y 5 yles of denaturation at 98 C for 10 s and annealing/extension at 7 C for min, followed y a final extension at 7 C for 5 min. SDM produts were leaned, DpnI digested and transformed into E. oli as previously desried. Funtional haraterisation of the himeri elongase proteins in Saharomyes erevisiae

7 Saharomyes erevisiae strain INVS1 was transformed with eah himeri onstrut for the prodution of reominant protein as previously desried (5). Reominant yeast expressing himeri elongase protein were supplemented with µm of 0:5n-3 (EPA) (Sapphire Biosiene, Waterloo, NSW, Australia) for h. Eah himeri protein was funtionally haraterised efore susequent onstruts were made. Data are expressed as the mean ± SD of inuations from three independent samples. Fatty aid analysis Total lipid was extrated from yeast ells and analysed y gas hromatography as previously desried (1). The amount of eah fatty aid was expressed as a perentage of the total amount of all fatty aids. This was done y expressing the peak area for an individual fatty aid as a perentage of the total peak area for all fatty aids. Statistial analysis One-way ANOVA with Tukey s post-ho test was performed using Graphpad Prism Downloaded from y guest, on Otoer 7, 018 version 5.03 for Windows (Graphpad Software, San Diego, CA, USA). Statistial signifiane was set at P<

8 RESULTS Sequene analysis of rat Elovl5 and Elovl The rat Elovl5 and Elovl proteins share 5% identity and inlude the strutural features harateristi of mirosomal fatty ayl elongases inluding seven transmemrane segments (TMS). Twenty three residues spanning TMS and TMS7 in Elovl5 and Elovl were identified to have lower identity, 3%, ompared to any similar region etween TMS1-5 (Fig. 1). Native Elovl5 and Elovl ativity Although the topi of this investigation is elongation, EPA was used as the sustrate eause it is ommon to oth enzymes and the that is elongated y Elovl is endogenously formed from EPA. EPA aumulation in the yeast ells expressing the rat Elovl5 or Elovl was proportional to the onentration of EPA added to the medium (data not shown). Elovl5 and Elovl synthesis of inreased proportionally with EPA sustrate onentration (Fig. A-B). However, Elovl further elongated the newly synthesised to :5n-3 (Fig. B) whereas Elovl5 did Downloaded from y guest, on Otoer 7, 018 not (Fig. A). Identifiation of Elovl residues involved in sustrate speifiity Twenty three residues from T01 to S3 that span Elovl TMS and TMS7 were replaed with the equivalent residues from Elovl5 to form himera 1 (Fig. 3). This resulted in a loss of the unique Elovl onversion of to :5n-3, ut retention of the aility to onvert EPA to (Fig. A). 8

9 To investigate whih ominations of residues were responsile for the to :5n-3 funtion, residues were progressively hanged ak to the original Elovl sequene. Initially, hanges were made to leave Elovl5 residues in eah TMS with the rationale that these would e adjaent if TMS and TMS7 formed a hannel for fatty aid sustrates. Ten of the twenty three Elovl5 residues that spanned the extraellular loop and the two adjaent memrane residues in eah TMS were hanged ak to Elovl residues V07-G1 in himera (Fig. 3). Chimeri protein displayed Elovl5-like ativity with elongation of EPA to ut no further (Fig. B). The restoration of a further three Elovl residues A0, C17 and L18 in himeri protein 3 resulted in :5n-3 synthesis eing partially returned (Figs. 3 and C). Levels of :5n-3 reahed.% of total fatty aids after 00 µm EPA supplementation (Fig. C) ompared to 3.8% :5n-3 with native Elovl (Fig. 3). In himeras and 5 a further three Elovl residues I19-Q1 or T03-S05 were restored in TMS7 or TMS, respetively (Fig. 3). In oth ases, this restored full Elovl to :5n-3 ativity with :5n-3 synthesis at 00 µm EPA supplementation reahing.1% and 3.% of total fatty aids, respetively (Figs. 3 and D-E). Downloaded from y guest, on Otoer 7, 018 The effet of Elovl5 point mutations on EPA ativity Results with himeri proteins 3, and 5 demonstrate that the residues important for native Elovl ativity inlude L0-A0 in TMS and C17-I19 in TMS7. These residues orrespond with Elovl5 S18-G0 in TMS and W31-Y33 in TMS7. Within these six residues, only Elovl L18/Elovl5 L3 is onserved. The effet of individual sustitutions of the other five Elovl5 residues with the orresponding Elovl residue was investigated (Fig. 5). The Elovl5 W31C mutant 9

10 showed a gain of Elovl-like to :5n-3 funtion (Fig. 5A) unlike the Elovl5 Y33I (Fig. 5B), S18L (Fig. 5C), C19S (Fig. 5D) and G0A (Fig. 5E) mutants whih retained Elovl5-like EPA to ativity, with an insignifiant amount of :5n-3 produed. The effet of Elovl point mutations on EPA ativity The sustitution of ysteine for tryptophan in the Elovl5 W31C mutant showed the importane of Elovl ysteine at position 17 for elongation of to :5n-3. When Elovl C17 was sustituted into the equivalent position in Elovl5 there was a restoration of :5n-3 synthesis (Fig. 5A). In the reverse mutant where Elovl5 W31 was sustituted into the equivalent position in Elovl, the aility to onvert to :5n-3 was lost ut EPA to syntheti apaility was retained (Fig. A). To further examine the role of tryptophan in the loss of Elovl elongation, Elovl point mutations were made with either the less spae-filling residue alanine or another ulky residue phenylalanine. The Elovl C17A (Fig. B) and Elovl C17F (Fig. C) mutants retained to :5n-3 ativity although at redued levels with :5n-3 reahing.% and 1.8%, respetively, after 00 µm EPA supplementation, ompared Downloaded from y guest, on Otoer 7, 018 to 3.8% with native Elovl (Fig. B). Comparing funtionally haraterised fish and mammalian Elovl5 and Elovl The region of 3 residues examined in himera 1 was ompared with the dedued Elovl5 and Elovl protein sequenes aross other mammals and fish (Fig. 7). There is only one position where a residue is onserved aross Elovl5 from all speies whih is different to the onserved residue aross Elovl from all speies and this is the 10

11 tryptophan at position 31 in rat Elovl5 whih is equivalent to the ysteine at position 17 in rat Elovl (Fig. 7). Downloaded from y guest, on Otoer 7,

12 DISCUSSION The initial reason for examining the TMS and TMS7 region of the rat Elovl5 and Elovl arose from a report that the TMS and TMS7 region of the yeast elongase, Surp, was responsile for the elongation of C 18 sustrates to C and the determination of the hain length (13). If this region is important for elongation ativity in the rat enzymes, inluding the different sustrate speifiities etween Elovl5 and Elovl, it is expeted that within the two sequenes there must e regions of homology whih enale oth proteins to elongate EPA and other regions of heterogeneity whih enale Elovl5 and Elovl to elongate SDA or, respetively. An alignment of the rat Elovl5 and Elovl proteins highlighted a 3 residue region of heterogeneity spanning TMS and TMS7. This region was targeted for involvement in sustrate speifiity due to its lower sequene identity ompared to the overall sequene. Confirmation that the TMS and TMS7 region was important for the differing sustrate speifiities etween Elovl5 and Elovl was provided y himera 1 in whih Downloaded from y guest, on Otoer 7, residues that span Elovl TMS and TMS7 were replaed with the equivalent residues from Elovl5. This resulted in a loss of the unique Elovl onversion of to :5n-3, ut with retention of the aility to onvert EPA to. This suess provided the platform for the sequential hanges in the Elovl5 insert to determine whih residues were important in restoring the Elovl funtionality of onverting EPA to and then to :5n-3. Chimeras and 5 demonstrated that five residues were potentially important, three in TMS and two in TMS7. Of these five residues, the point mutations revealed that it was the C17 residue of Elovl that was ritial 1

13 for elongation of at the level oserved in the native protein. A further finding is that the loss of to :5n-3 ativity in Elovl C17W appears to e aused at least in part y the inlusion of a tryptophan residue, whih is at the equivalent position in Elovl5, and not simply due to the loss of the ysteine residue at this position. Cysteine is a less spae-filling residue than tryptophan whih may failitate the entry of further into the transmemrane hannel. However, the same sustitution with another hydrophoi residue suh as phenylalanine, whih ontains a enzyl side hain similar to tryptophan or the struturally simple alanine, retained to :5n-3 ativity, although at redued levels. The aility of Elovl to elongate may e due to the effet of C17 in TMS7 on the struture of Elovl. An alignment of the dedued Elovl5 and Elovl protein sequenes from other funtionally haraterised mammals and fish supports the essentiality of ysteine at the equivalent position aross all Elovl proteins (Fig. 7). Likewise a tryptophan is found at the equivalent position aross all 1 of the Elovl5 sequenes used in the alignment (Fig. 7). Downloaded from y guest, on Otoer 7, 018 Although himera 1 and Elovl C31W resulted in a loss of ativity making the enzymati ativity of these proteins more Elovl5-like, these proteins did not gain signifiant Elovl5-like SDA ativity (data not shown). Similarly, the gain of ativity y Elovl5 W31C did not result in a loss of Elovl5-like SDA ativity (data not shown). These findings suggest that the residues responsile for SDA sustrate speifiity are not within TMS and TMS7 ut instead in a separate region of Elovl5. 13

14 The opposite himeri onstrut was made y replaing the 3 residues from I15 to G37 that span Elovl5 TMS and TMS7 with the equivalent from Elovl. Interestingly, this himeri protein was inative when expressed in yeast and no longer ale to onvert SDA or EPA (data not shown). A similar finding was reported in the fungi elongases when himeri proteins of PirELO and PinELO were made. The inlusion of a region of PirELO residues in PinELO resulted in a gain of EPA sustrate speifiity whereas the reiproal himera resulted in an inative PirELO whih was no longer ale to onvert GLA or EPA (15). We have reported that the hiken Elovl5 has some aility to elongate (9). This is unlike the Elovl5 enzymes of rat (5, 17), human (18) and most, ut not all fish (, 8, 1, 19, 0). The urrent study does not identify sequene differenes etween the hiken and rat Elovl5 whih may explain their different ailities to elongate. Also, it does not identify sequene variaility whih ould explain the higher ativity of the hiken Elovl5 whih onverts 0% to :5n-3 ompared with Elovl5 onversion ativities of 5-9% in sea ream, zerafish, oia and Atlanti luefin tuna (8, 10, 11, 1). The sites within these Elovl5 enzymes that onfer Downloaded from y guest, on Otoer 7, 018 elongation aility may not e within the transmemrane regions examined in this study with rat enzymes. The results of this study provide a starting point for further examination of the differing ailities of Elovl5 and Elovl to elongate and the differing ailities of Elovl5 enzymes in different speies to elongate. A omprehensive understanding of the moleular differenes responsile for these differing ativities ould allow sequene data to e used to assess the aility of a speies or different 1

15 reeds of a domesti speies to elongate to :5n-3, a ritial and perhaps ratelimiting reation for DHA synthesis. Downloaded from y guest, on Otoer 7,

16 REFERENCES 1. Burdge, G. C., A. E. Jones, and S. A. Wootton. 00. Eiosapentaenoi and doosapentaenoi aids are the prinipal produts of alpha-linoleni aid metaolism in young men*. Br J Nutr 88: James, M. J., V. M. Ursin, and L. G. Cleland Metaolism of stearidoni aid in human sujets: omparison with the metaolism of other n-3 fatty aids. Am J Clin Nutr 77: Fu, Z., and A. J. Sinlair Inreased alpha-linoleni aid intake inreases tissue alpha-linoleni aid ontent and apparent oxidation with little effet on tissue doosahexaenoi aid in the guinea pig. Lipids 35: Bowen, R. A., and M. T. Clandinin High dietary 18:3n-3 inreases the 18:3n-3 ut not the :n-3 ontent in the whole ody, rain, skin, epididymal fat pads, and musles of sukling rat pups. Lipids 35: Gregory, M. K., R. A. Gison, R. J. Cook-Johnson, L. G. Cleland, and M. J. James Elongase reations as ontrol points in long-hain polyunsaturated fatty aid synthesis. PLoS One : e9. Downloaded from y guest, on Otoer 7, 018. Tu, W. C., B. S. Muhlhausler, M. J. James, D. A. Stone, and R. A. Gison. 01. An alternative n-3 fatty aid elongation pathway utilising 18:3n-3 in arramundi (Lates alarifer). Biohem Biophys Res Commun 3: Tu, W. C., B. S. Muhlhausler, M. J. James, D. A. Stone, and R. A. Gison Dietary alpha-linoleni aid does not enhane aumulation of omega-3 long-hain polyunsaturated fatty aids in arramundi (Lates alarifer). Comp Biohem Physiol B Biohem Mol Biol 1:

17 8. Agaa, M. K., D. R. Toher, X. Zheng, C. A. Dikson, J. R. Dik, and A. J. Teale Cloning and funtional haraterisation of polyunsaturated fatty aid elongases of marine and freshwater teleost fish. Comp Biohem Physiol B Biohem Mol Biol 1: Gregory, M. K., M. S. Geier, R. A. Gison, and M. J. James Funtional haraterization of the hiken fatty aid elongases. J Nutr 13: Morais, S., G. Mourente, A. Ortega, J. A. Toher, and D. R. Toher Expression of fatty ayl desaturase and elongase genes, and evolution of DHA:EPA ratio during development of unfed larvae of Atlanti luefin tuna (Thunnus thynnus L.). Aquaulture 313: Zheng, X., Z. Ding, Y. Xu, O. Monroig, S. Morais, and D. R. Toher Physiologial roles of fatty ayl desaturases and elongases in marine fish: Charaterisation of DNAs of fatty ayl desaturase and elovl5 elongase of oia (Rahyentron anadum). Aquaulture 90: Guillou, H., D. Zadrave, P. G. Martin, and A. Jaosson The key roles of elongases and desaturases in mammalian fatty aid metaolism: Insights from transgeni mie. Prog Lipid Res 9: Downloaded from y guest, on Otoer 7, Deni, V., and J. S. Weissman A moleular aliper mehanism for determining very long-hain fatty aid length. Cell 130: Eiamsa-Ard, P., A. Kanjana-Opas, E. B. Cahoon, P. Chodok, and S. Kaewsuwan Two novel Physomitrella patens fatty aid elongases (ELOs): identifiation and funtional haraterization. Appl Miroiol Biotehnol 97:

18 15. Vrinten, P. L., T. Hoffman, J. Bauer, and X. Qiu Speifi protein regions influene sustrate speifiity and produt length in polyunsaturated fatty aid ondensing enzymes. Biohemistry 9: Gregory, M. K., V. H. See, R. A. Gison, and K. A. Shuller Cloning and funtional haraterisation of a fatty ayl elongase from southern luefin tuna (Thunnus maoyii). Comp Biohem Physiol B Biohem Mol Biol 155: Inagaki, K., T. Aki, Y. Fukuda, S. Kawamoto, S. Shigeta, K. Ono, and O. Suzuki. 00. Identifiation and expression of a rat fatty aid elongase involved in the iosynthesis of C18 fatty aids. Biosi Biotehnol Biohem : Leonard, A. E., E. G. Boik, J. Dorado, P. E. Kroeger, L. T. Chuang, J. M. Thurmond, J. M. Parker-Barnes, T. Das, Y. S. Huang, and P. Mukerji Cloning of a human DNA enoding a novel enzyme involved in the elongation of long-hain polyunsaturated fatty aids. Biohem J 350 Pt 3: Hastings, N., M. K. Agaa, D. R. Toher, X. Zheng, C. A. Dikson, J. R. Downloaded from y guest, on Otoer 7, 018 Dik, and A. J. Teale. 00. Moleular loning and funtional haraterization of fatty ayl desaturase and elongase DNAs involved in the prodution of eiosapentaenoi and doosahexaenoi aids from alpha-linoleni aid in Atlanti salmon (Salmo salar). Mar Biotehnol (NY) : Meyer, A., H. Kirsh, F. Domergue, A. Aadi, P. Sperling, J. Bauer, P. Cirpus, T. K. Zank, H. Moreau, T. J. Rosoe, U. Zahringer, and E. Heinz. 00. Novel fatty aid elongases and their use for the reonstitution of doosahexaenoi aid iosynthesis. J Lipid Res 5:

19 1. Agaa, M., D. R. Toher, C. A. Dikson, J. R. Dik, and A. J. Teale. 00. Zerafish DNA enoding multifuntional Fatty Aid elongase involved in prodution of eiosapentaenoi (0:5n-3) and doosahexaenoi (:n-3) aids. Mar Biotehnol (NY) : Downloaded from y guest, on Otoer 7,

20 FIGURE LEGENDS Fig. 1. A dedued amino aid sequene alignment of Elovl5 and Elovl from rat. Elovl5 amino aid numering is shown aove the alignment and Elovl numering shown elow the alignment. Identity/similarity shading was ased on the Gonnet series matrix produed y ClustalX where primary lak shading indiates idential residues and seondary and tertiary grey shading indiates similar residues with an 80% and 0% ut off, respetively. Seven predited transmemrane spanning domains were predited using and are shown with a solid lined ox and a region of lower identity etween transmemrane segments and 7 is shown with a dashed lined ox. Fig.. Elongation of EPA y rat Elovl5 (A) and Elovl (B). Values represent the means ± S.D. of tripliate inuations. Values with different symols are signifiantly different from eah other. The inserts show the Elovl5 or Elovl amino aid sequene of TMS, TMS7 and the extraellular loop. Downloaded from y guest, on Otoer 7, 018 Fig. 3. Rat Elovl/Elovl5 himeri proteins. Amino aid numering in the himeras is aording to the Elovl residue position. The tale shows the amount of and :5n-3 in yeast expressing himeri proteins after inuation with 00 µm EPA. Fig.. Elongation of EPA y himeri rat elongase proteins. Chimera 1 (A), himera (B), himera 3 (C), himera (D), himera 5 (E). Values represent the means ± S.D. of tripliate inuations. Values with different symols are signifiantly different 0

21 from eah other. The inserts show the Elovl5 residues in lue and Elovl residues in red within eah himeri onstrut aross TMS, TMS7 and the extraellular loop. Fig. 5. Elongation of EPA y rat Elovl5 ontaining amino aid point mutations Elovl5 W31C (A), Elovl5 Y33I (B), Elovl5 S18L (C), Elovl5 C19S (D) or Elovl5 G0A (E). Values represent the means ± S.D. of tripliate inuations. Values with different symols are signifiantly different from eah other. The inserts show the Elovl5 amino aid sequene of TMS, TMS7 and the extraellular loop in lue with the Elovl amino aid point mutation in red. Fig.. Elongation of EPA y rat Elovl ontaining amino aid position 17 point mutations Elovl C17W (A), Elovl C17A (B) or Elovl C17F (C). Values represent the means ± S.D. of tripliate inuations. Values with different symols are signifiantly different from eah other. The inserts show the Elovl amino aid sequene of TMS, TMS7 and the extraellular loop in red, the Elovl5 amino aid point mutation in lue and alternative amino aid point mutations in lak. Downloaded from y guest, on Otoer 7, 018 Fig. 7. A dedued amino aid sequene alignment of mammalian and fish Elovl5 and Elovl. Identity/similarity shading was ased on the Gonnet series matrix produed y ClustalX where primary lak shading indiates idential residues and seondary and tertiary grey shading indiates similar residues with an 80% and 0% ut off, respetively. The onserved tryptophan in all Elovl5 sequenes and ysteine in all Elovl sequenes is indiated with an arrow. 1

22 Figure 1 Downloaded from y guest, on Otoer 7, 018

23 % of total fatty aid % of total fatty aid Figure A Elovl5 B Elovl :5n-3 a a a Downloaded from y guest, on Otoer 7, 018 3

24 Figure 3 Downloaded from y guest, on Otoer 7, 018

25 % of total fatty aid % of total fatty aid % of total fatty aid % of total fatty aid % of total fatty aid Figure A Chimera 1 B Chimera a a C E Chimera 3 a a Chimera 5 :5n-3 D Chimera :5n-3 a a Downloaded from y guest, on Otoer 7, 018 :5n-3 a a a

26 % of total fatty aid % of total fatty aid % of total fatty aid % of total fatty aid % of total fatty aid Figure 5 A Elovl5 W31C B Elovl5 Y33I 10 8 :5n :5n-3 aa a a a C E 10 8 Elovl5 S18L Elovl5 G0A :5n-3 a a D 10 8 Elovl5 C19S a 0 a :5n-3 Downloaded from y guest, on Otoer 7, :5n-3 a a

27 % of total fatty aid % of total fatty aid % of total fatty aid Figure A Elovl C17W 8 :5n-3 B C a a 0 a Elovl C17A aa Elovl C17F :5n-3 Downloaded from y guest, on Otoer 7, :5n-3 a a

28 Figure 7 Downloaded from y guest, on Otoer 7, 018 8