Distinct roles of endoplasmic reticulum cytochrome b 5 and fused cytochrome b 5-like domain for rat 6-desaturase activity

The 6-desaturase catalyzes key steps in longchain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat 6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of 6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and 6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for 6-desaturase activity. —Guillou, H., S. D’Andrea, V. Rioux, R. Barnouin, S. Dalaine, F. Pedrono, S. Jan, and P. Legrand. Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat 6-desaturase activity. J. Lipid Res. 2004. 45: 32–40. Supplementary key words FADS2 • polyunsaturated fatty acid biosynthesis • hexadecenoic acid Long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) play pivotal roles in a variety of biological functions (1). In animals, some of the daily needs in longchain PUFAs are fulfilled from the diet. However, most of the long-chain PUFAs found in animal tissues are derived from the biosynthetic pathway involving elongations, 6desaturation, and 5-desaturation for conversion of essential fatty acid precursors (C18:2n-6 and C18:3n-3) to their respective 20and 22-carbon polyenoic products. None of the desaturases involved in this biosynthetic pathway have been reproducibly purified, and their structure remains to be characterized. The only animal desaturase whose structure is known is the 9-desaturase (2). This enzyme is part of a multienzyme system present in the endoplasmic reticulum and is composed of 9-desaturase, NADH cytochrome b5 reductase, and cytochrome b5. In the process of double bond formation, the membrane-bound cytochrome b5 transfers electrons by lateral diffusion from NADH cytochrome b5 reductase to the 9 fatty acid desaturase (2). Although the first mammalian 9-desaturase was cloned almost 20 years ago (3), mammalian desaturases involved in PUFA biosynthetic pathways, i.e., 6and 5-desaturases, have been cloned more recently (4–8). Comparison of their respective amino acid sequences shows one major difference between 9-desaturase and 6and 5-desaturases: an N-terminal cytochrome b5-like domain is present in 6and 5-desaturases but not in 9-desaturase. Numerous cytochrome b5-like domains have been identified in various desaturases from yeast, plants, and animals (9). This remarkable characteristic raises the possibility that NADH cytochrome b5 reductase transfers electrons to the catalytic site of these cytochrome b5 fusion desaturases directly via the cytochrome b5-like domain and does not require an independent cytochrome b5. The presence of such cytochrome b5-like domains in desaturase proteins is likely to have originated from a fusion with an ancestral cytochrome b5 gene that may have conferred some evolutionarily selectable advantage. Although these cytochrome Abbreviations: FCS, fetal calf serum; GC, gas chromatography. 1 To whom correspondence should be addressed. e-mail: philippe.legrand@agrorennes.educagri.fr The online version of this article (available at http://www.jlr.org) contains an additional three figures. Manuscript received 1 August 2003 and in revised form 1 October 2003. Published, JLR Papers in Press, October 16, 2003. DOI 10.1194/jlr.M300339-JLR200 by gest, on O cber 3, 2017 w w w .j.org D ow nladed fom 0.DC1.html http://www.jlr.org/content/suppl/2004/01/23/M300339-JLR20 Supplemental Material can be found at:

Long-chain polyunsaturated fatty acids (PUFAs) such as arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) play pivotal roles in a variety of biological functions (1).In animals, some of the daily needs in longchain PUFAs are fulfilled from the diet.However, most of the long-chain PUFAs found in animal tissues are derived from the biosynthetic pathway involving elongations, ⌬ 6-desaturation, and ⌬ 5-desaturation for conversion of essential fatty acid precursors (C18:2n-6 and C18:3n-3) to their respective 20-and 22-carbon polyenoic products.
None of the desaturases involved in this biosynthetic pathway have been reproducibly purified, and their structure remains to be characterized.The only animal desaturase whose structure is known is the ⌬ 9-desaturase (2).This enzyme is part of a multienzyme system present in the endoplasmic reticulum and is composed of ⌬ 9-desaturase, NADH cytochrome b5 reductase, and cytochrome b5.In the process of double bond formation, the membrane-bound cytochrome b5 transfers electrons by lateral diffusion from NADH cytochrome b5 reductase to the ⌬ 9 fatty acid desaturase (2).
Numerous cytochrome b5-like domains have been identified in various desaturases from yeast, plants, and animals (9).This remarkable characteristic raises the possibility that NADH cytochrome b5 reductase transfers electrons to the catalytic site of these cytochrome b5 fusion desaturases directly via the cytochrome b5-like domain and does not require an independent cytochrome b5.The presence of such cytochrome b5-like domains in desaturase proteins is likely to have originated from a fusion with an ancestral cytochrome b5 gene that may have conferred some evolutionarily selectable advantage.Although these cytochrome b5 fusion domains have diverged significantly, a typical HPGG motif has been conserved.This particular sequence forms an accessible heme binding core of the cytochrome b5-like domain (10).Among desaturases fused to a cytochrome b5-like domain, the cytochrome b5 domain has been demonstrated to be essential for borage ⌬ 6-desaturase (11) and a yeast ⌬ 9-acyl-CoA-desaturase (12).
In this study, we compared, in COS-7 cells, the activity of recombinant wild-type rat ⌬ 6-desaturase with the activity of mutated recombinant enzymes in which the typical cytochrome b5 53 HPGG 56 motif has been mutated or deleted.We also investigated in COS-7 cells the role of coexpressed microsomal cytochrome b5 in ⌬ 6-desaturase activity and its putative capacity to compensate for the essentialness of the 53 HPGG 56 motif in the ⌬ 6-desaturase function reported here.
A plasmid coding for rat cytochrome b5 was constructed for expression in mammalian cells and is referred to as pcDNA3/ cytb5.From the published (20) rat cytochrome b5 sequence (GenBank accession number D13205), oligonucleotide primers were designed to PCR amplify the entire coding sequence, with its stop codon using the high-fidelity Pfu polymerase from Promega (Lyon, France).The forward primer (5 Ј -CAATGGATC-C ATG CCGGCCCACATGC-3 Ј ) included the translation start codon (boldface) and the Bam HI restriction site (underlined).The reverse primer (5 Ј -CGTGCTCGAGC TCA GCTACTCTTGT-GGCT-3 Ј ) contained the translation stop codon (boldface) and the Xho I restriction site (underlined).The PCR product amplified from rat liver cDNA was treated with Bam HI and Xho I before cloning into pcDNA3 (Invitrogen, San Diego, CA).The Hind III-Sal I fragment containing the full-length rat cytochrome b5 cDNA was subcloned in frame from pcDNA3 into pCMV-HAHA.This construction is referred to as pCMV-HAHA/cytb5 and allows the expression of a cytochrome b5 fused N-terminally to a double hemagglutinin (HA) epitope.
A plasmid coding for a C-terminally myc-tagged ⌬ 6-desaturase was constructed using pCMV for expression in mammalian cells and is referred to as pCMV/ ⌬ 6myc.From the published rat ⌬ 6desaturase sequence (5) (GenBank accession number AB021980), oligonucleotide primers were designed to amplify, by PCR, the entire coding sequence with a deleted stop codon.The forward primer (5 Ј -CAGTGGATCC ATG GGGAAGGGAGGTA-3 Ј ) included the translation start codon (italics) and the Nco I restriction site (underlined).The reverse primer (5 Ј -TGTGCGGCCGC TTTGT-GGAGGTAGGCATCC -3 Ј ) corresponded to the C-terminal sequence of the protein (italics) without its stop codon and a Not I site (underlined).The PCR product amplified from rat liver cDNA was treated with Nco I and Not I before cloning into pCMV/ myc/cyto (Invitrogen).
Mutagenesis of the 53 HPGG 56 motif in the N-terminal cytochrome b5 domain of rat ⌬ 6-desaturase was performed using a site-directed mutagenesis kit (QuickChange; Stratagene, Amsterdam, The Netherlands) according to the manufacturer's protocol.Three sets of two mutagenic primers were designed ( Table 1 ).In each set, both primers are complementary to the opposite strands of pCMV/ ⌬ 6 and insert the desired mutation or deletion.These primers were used to delete the 53 HPGG 56  The mutagenic codon is indicated in boldface.The codon deletions are indicated (X).

Cell culture and transfection
COS-7 cells were routinely maintained at ‫%05ف‬ confluence and were cultured in DMEM containing 10% FCS, 50 IU/ml penicillin, and 50 g/ml streptomycin.The cells were split 1 day before transfection to 30% confluence and transfected the next day using the Easyject Plus electroporator (Equibio, Monchelsea, UK) according to the manufacturer's instructions.Briefly, 10 6 COS-7 cells in 0.8 ml of DMEM were mixed with 30 g of purified plasmid, electroporated at 250 V and 1,500 F with unlimited resistance, and seeded on a 10 cm dish containing culture medium.

Immunofluorescence
Coverslips containing the paraformaldehyde-fixed cells transfected with pCMV/⌬6myc were washed in PBS (150 mM NaCl and 5 mM Na phosphate, pH 7.4) and preincubated for 10 min on a drop of blocking buffer containing Triton X-100 (PBS containing 0.5% BSA and 0.1% Triton X-100).The cells were extensively washed with blocking buffer and incubated for 30 min with the primary antibody (monoclonal anti-Myc) in blocking buffer (1:2 dilution).After extensive washes in blocking buffer, the cells were incubated for 30 min with the fluorescent secondary antibody (anti-mouse IgG FITC; Sigma) diluted in blocking buffer (1:200 dilution).The coverslips were again washed extensively in blocking buffer and once in PBS, mounted in Tris-HCl (0.5 M, pH 8.5) containing 70% glycerol, and observed under a Leica DMRB microscope equipped for epifluorescence.

Incubation of transfected COS-7 cells with fatty acid albuminic complex
The functionality of the expressed protein was investigated by incubating the transfected COS-7 cells with different fatty acid albuminic complexes.Each fatty acid was saponified by incubation for 30 min at 70ЊC with 2 M KOH in ethanol.The resulting fatty Fig. 1.A: Expression of myc-tagged rat ⌬6-desaturase was assessed by Western blotting of transiently transfected COS-7 cell lysates using anti-rat ⌬6-desaturase sera.B: Localization of myctagged ⌬6-desaturase expressed in COS-7 cells.COS-7 cells were transiently transfected with pCMV/⌬6myc, fixed, permeabilized, and stained for myc epitope using FITC-labeled secondary antibodies.No signal was detected in nontransfected cells incubated under the same conditions (data not shown).

Fig. 2.
In vitro desaturation of [1-14 C]C18:3n-3 measured in COS-7 cell homogenates not transfected (control) or containing wild-type (transfected with pCMV/⌬6) and mutated forms (transfected with pCMV/⌬6ϪH 53 A, pCMV/⌬6ϪH 53 , or pCMV/⌬6Ϫ 53 HPGG 56 ) of rat ⌬6desaturase.Desaturase activity was calculated from the level of [1-14 C]C18:4n-3 produced, normalized to ␤-galactosidase activity, and expressed as means Ϯ standard deviations between triplicates.Expression of wild-type and mutated forms of desaturase was assessed by Western blotting of cell lysates.Anti-actin level was assessed by Western blotting as a loading control.This result is representative of three independent experiments.The asterisk indicates a significant difference compared with the control by Student's t-test (P Ͻ 0.05).
acid salt was dissolved at pH 10 in DMEM containing 1% (w/v) BSA.After 15 min of sonication followed by 5 h of shaking, the pH was adjusted to 7.3.FCS was added (10%, v/v), and the final fatty acid concentration of the incubation medium was 0.2 mM unless stated otherwise.At 3 h after transfection, the incubation of COS-7 cells was initiated by replacing the culture medium with 20 ml of the fatty acid-containing medium per 10 cm dish.Incubation was performed for 24 h at 37ЊC in 5% CO 2 atmosphere.

Fatty acid analysis
COS-7 cells were washed twice with ice-cold PBS (150 mM NaCl and 5 mM Na phosphate, pH 7.4) and scraped into PBS.After centrifugation, the cell pellet was resuspended in PBS and sonicated at 20 W for 5 s.The protein content of the cell homogenate was determined by a modified Lowry procedure (21).Cellular lipids were extracted with hexane-isopropanol (3:2; v/v) as described previously (22).After saponification, fatty acids were methylated with boron trifluoride (14% in methanol) at 70ЊC for 30 min.Fatty acid methyl esters were extracted with pentane and analyzed by gas chromatography using an Agilent Technologies 6890N (Bios Analytique, Toulouse, France) with a split injector (1:20) at 250ЊC and a bonded silica capillary column (30 m ϫ 0.25 mm internal diameter; BPX 70; SGE, Villeneuve-St-Georges, France) with a stationary phase of 70% cyanopropylpolysilphenylene-siloxane (0.25 m film thickness).Helium was used as the gas vector (average velocity, 24 cm/s).The column temperature program started at 150ЊC, was ramped at 2ЊC/min to 220ЊC, and was held at 220ЊC for 10 min.The flame ionization detector temperature was 250ЊC.Identification of fatty acid methyl ester peaks was based on retention times obtained for methyl esters prepared from fatty acid standards.

Enzyme assay
Cell homogenates were prepared as described above at 48 h after transfection.Desaturase activity was assayed in a 1 ml mixture containing 100 l of cell homogenate (5-8 mg protein/ml), Fig. 3. Gas chromatography(GC) analysis of fatty acid methyl ester from COS-7 cells (A), COS-7 cells incubated with C18:3n-3 (B), or COS-7 cells transiently transfected with pCMV/⌬6 (C), pCMV/⌬6ϪH 53 (D), pCMV/ ⌬6ϪH 53 A (E), or pCMV/⌬6Ϫ 53 HPGG 56 (F) incubated with C18:3n-3.COS-7 cells were transfected or not and subsequently cultivated or not for 24 h with albuminbound C18:3n-3 (200 M).Then, the cells were washed extensively with PBS and cellular fatty acids were prepared for GC analysis of fatty acid methyl esters as described in Materials and Methods.The results presented are representative of three independent experiments.150 mM phosphate buffer (pH 7.2), 6 mM MgCl 2 , 7.2 mM ATP, 0.54 mM CoA, and 0.8 mM NADH.The reaction was started by adding 60 nmol of [1-14 C]18:3n-3 (52 mCi/mmol) and stopped with 1 ml of 2 M KOH in ethanol after 1 h of incubation at 37ЊC.To assess the substrate quality, a control assay was also run by stopping the reaction before adding the substrate.Fatty acid saponification was performed at 70ЊC for 30 min.After acidification, the fatty acids were extracted with diethylether, converted to fatty acid naphthacyl esters, and separated by HPLC as described previously (23).Collected fractions were subjected to liquid scintillation counting (Packard Tri-Carb 1600 TR, Meriden, CT).Desaturase activities were normalized for transfection efficiency by measuring the ␤-galactosidase activity corresponding to 3 g of a cotransfected ␤-galactosidase-expressing vector (pCMV/␤-Gal).

Immunoprecipitation and immunoblotting
Two days after transfection, cells were lysed at 4ЊC in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 mM PMSF, and 10 g/ ml aprotinin).Lysates were subjected to 12 h of immunoprecipitation with 1 g of either monoclonal anti-Flag M2 (Sigma) or polyclonal anti-HA Y11 (Santa Cruz Biotechnologies, Le Perrayen-Yvelines, France) followed by adsorption to Sepharose-coupled protein G (Sigma) for 3 h.Immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting.For determination of total protein levels, aliquots of cell lysates were also subjected to direct immunoblotting.
Reduced protein samples were analyzed by SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany).To measure wild-type and mutant ⌬6-desaturase expression, the anti ⌬6-desaturase serum S1 targeting the C-terminal region of the protein was used at a 1:2,000 dilution, as described previously (13).To measure cytochrome b5 expression, anti-cytochrome b5 (24) was used at a 1:200 dilution.Anti-HA was used at a 1:200 dilution, anti-Flag was used at a 1:300 dilution, and anti-actin (Sigma) was used at a 1:100 dilution.The secondary antibody was a peroxidase-conjugated anti-rabbit IgG (Sigma) or a peroxidaseconjugated anti-mouse IgG (Sigma).Saturation and incubation with antibodies were performed for 90 min in TBS (20 mM Tris-HCl and 150 mM NaCl, pH 7.4) containing 0.05% Tween-20 and 5% nonfat dry milk.Washes were performed in TBS containing 0.05% Tween-20.Peroxidase activity was revealed using ECL Plus reagent according to the manufacturer's instructions (Amersham Biosciences, Uppsala, Sweden) and scanned with the Molecular Dynamics Storm (Amersham Biosciences).

Expression and activity of wild-type and mutated rat ⌬6-desaturases in COS-7 cells
The wild-type (⌬6) and the three mutated forms of rat ⌬6-desaturases with deletion of the 53 HPGG 56 motif (⌬6Ϫ 53 HPGG 56 ), deletion of H 53 (⌬6ϪH 53 ), or substitution of an alanine for H 53 (⌬6ϪH 53 A) expressed in COS-7 cells were analyzed by Western blotting.A serum targeting the C-terminal region of the protein ( 13), which has not been modified by site-directed mutagenesis, was used to probe the membranes.Western blotting revealed that the wild type and the three mutated forms of rat ⌬6-desaturase were similarly expressed in COS-7 cells (Fig. 2).The deletion of the 53 HPGG 56 motif or of H 53 , or the substitution of an alanine for H 53 did not alter rat ⌬6-desaturase expression in this cell line.
The in vitro ⌬6-desaturase assay performed on the COS-7 cell lysates corresponding to the samples used for Western blotting showed a dramatic increase in ⌬6-desaturase activity only in cells expressing the wild-type enzyme, compared with nontransfected cells (Fig. 2).The deletion of the 53 HPGG 56 motif or of H 53 , or the substitution of an alanine for H 53 suppressed the activity of ⌬6-desaturase as measured in vitro.

Effect of rat cytochrome b5 on the activity of wild-type and mutated rat ⌬6-desaturases in COS-7 cells
The microsomal form of rat cytochrome b5 was cotransfected with the wild-type or mutated forms of ⌬6-desaturase in COS-7 cells.The expression of ⌬6-desaturases and cytochrome b5 was controlled by Western blotting (Fig. 4).Then, relative ⌬6-desaturase activities were measured in vitro on the cell lysates.This assay showed that overexpression of rat microsomal cytochrome b5 cannot compensate for the effect of the deletions or the mutation in the 53 HPGG 56 motif of the rat ⌬6-desaturase cytochrome b5 domain (Fig. 4).Interestingly, coexpression of rat endoplasmic reticulum cytochrome b5 with wild-type ⌬6desaturase markedly increased (2.2-fold) the ⌬6-desaturase activity, compared with ⌬6-desaturase alone.

Interaction between wild-type or mutated ⌬6-desaturase and cytochrome b5
The possibility that ⌬6-desaturase may interact with cytochrome b5 was investigated in COS-7 cells.Immunoprecipitation of lysates from transfected COS-7 cells with an antibody directed against Flag-tagged ⌬6-desaturase revealed the presence of HA-tagged cytochrome b5 (Fig. 5).Similarly, when immunoprecipitation was performed with an antibody directed against HA-tagged cytochrome b5, the Flag-tagged ⌬6-desaturase was coimmunoprecipitated. Interestingly, Flag-tagged ⌬6-desaturase lacking the HPGG motif also coimmunoprecipitated with HA-tagged cytochrome b5, suggesting that the 53 HPGG 56 motif of the desaturase is not required for interaction between ⌬6-desaturase and cytochrome b5.

DISCUSSION
This study presents the localization in an animal cell line of rat ⌬6-desaturase in the endoplasmic reticulum (Fig. 1) and provides the first evidence for the requirement of the 53 HPGG 56 motif, and at least H 53 in this motif, in the cytochrome b5-like domain of this enzyme.As the mutated proteins had no activity when expressed at levels similar to those in the wild type, we concluded that the domain is important for the protein activity but not for its expression or stability (Fig. 2).Moreover, our data showed that this motif, and particularly H 53 , plays a critical role in ⌬6-desaturase activity in its different substrates (Fig. 3, Table 2).This observation is consistent with the previously Fig. 5.The rat ⌬6-desaturase 53 HPGG 56 motif is not required for association with cytochrome b5.COS-7 cells were transfected with p3ϫFlag/⌬6 or p3ϫFlag/⌬6-HPGG in the absence or presence of pCMV-HAHA/ cytb5.Cell lysates were subjected to immunoprecipitation with anti-Flag antibody and anti-hemagglutinin (HA) antibody.The Flag-tagged desaturase immunoprecipitation was visualized with Flag immunoblotting (IP: ␣-Flag, Blot: ␣-Flag), and the HA-tagged cytochrome b5 immunoprecipitation was visualized with HA immunoblotting (IP: ␣-HA, Blot: ␣-HA).The association of HAtagged cytochrome b5 with Flag-tagged desaturases was visualized with HA immunoblotting (IP: ␣-Flag, Blot: ␣-HA) and with Flag immunoblotting (IP: ␣-HA, Blot: ␣-Flag).As controls, cell lysates were also subjected to immunoblotting with anti-Flag (Blot: ␣-Flag), anti-HA (Blot: ␣-HA), and anti-actin (Blot: actin).reported essentialness of H 41 in borage ⌬6-desaturase (11) and with the role of the cytochrome b5-like domain in yeast ⌬9-desaturase activity (12).
The COS-7 cells are unlikely to be deficient in cytochrome b5.For example, rat ⌬9-desaturase, which requires cytochrome b5 to function, is active when expressed in COS7 cells (15).This suggests that endogenous cytochrome b5, constitutively present in COS-7 cells, cannot rescue the activity of a ⌬6-desaturase whose cytochrome b5-like domain has been mutated or deleted.To further address this hypothesis, we measured ⌬6-desaturase activities in COS-7 cells transiently transfected with distinct forms of ⌬6-desaturase in the presence or absence of coexpressed rat cytochrome b5.Similarly, we showed that rat cytochrome b5 coexpressed in this cell line did not rescue the activity of mutated forms of ⌬6-desaturase (Fig. 4).Thus, neither endogenous microsomal cytochrome b5 nor coexpressed rat cytochrome b5 could rescue the activity of mutated ⌬6-desaturases.
The major role of the rat ⌬6-desaturase cytochrome b5-like domain may have led to the speculation that the enzyme can function independently of free microsomal cytochrome b5.However, coexpression of microsomal rat cytochrome b5 with rat ⌬6-desaturase is necessary for an optimal PUFA desaturation in yeast (17).Thus, the role of microsomal cytochrome b5 in the process of ⌬6-desaturation could not be dismissed.Consistent with this proposal, we showed that microsomal cytochrome b5 stimulated ⌬6desaturase activity when coexpressed in a mammalian cell line (Fig. 4).
Because the 53 HPGG 56 region of the rat ⌬6-desaturase cytochrome b5-like domain may represent an important motif for the structure of the protein and its putative interaction with other proteins, we tested whether ⌬6-desaturase or ⌬6-desaturase with complete deletion of the 53 HPGG 56 sequence interacts with cytochrome b5 in COS-7 cells.When coexpressed in COS-7 cells, wild-type ⌬6desaturase interacted with cytochrome b5 (Fig. 5).This protein-protein interaction may contribute to the effect of cytochrome b5 on ⌬6-desaturase activity.Interestingly, we observed that the complete deletion of the 53 HPGG 56 motif did not alter the interaction between ⌬6-desaturase and cytochrome b5 (Fig. 5), providing evidence that this motif is not necessary for interaction between these two proteins, whereas cytochrome b5 could not rescue the activity of mutated forms of ⌬6-desaturases (Fig. 4).
Therefore, the different results described here assess the important role of both the ⌬6-desaturase cytochrome b5-like domain and the microsomal cytochrome b5 in the process of ⌬6-desaturation.This study also shows that microsomal cytochrome b5 cannot compensate for the essential role of the highly conserved 53 HPGG 56 motif in the rat ⌬6-desaturase cytochrome b5-like domain.The precise role of free cytochrome b5 in ⌬6-desaturase activity should be further defined.It would be interesting to investigate further the cytochrome b5-⌬6-desaturase interaction and the stimulatory effect of cytochrome b5 using models with greater physiological expression of both proteins.Whether cytochrome b5 contributes to an electron transfer required for ⌬6-desaturase activity remains to be elucidated.As has been shown for cytochrome P450 monooxygenase (25), the possibility that cytochrome b5 may not function as an electron transfer component in the ⌬6-desaturase enzymatic system could be considered.Together, these results suggest essential and distinct roles for free cytochrome b5 and the fused cytochrome b5-like domain in governing ⌬6-desaturase activity.
by guest, onOctober 13, 2017 used for PCR amplification with oligonucleotide primers before subcloning into p3 ϫ Flag (Sigma).The forward primer (5 Ј -GACT-GAAGCTT ATG GGGAAGGGAGGTA-3 Ј ) included the translation start codon (italics) and a HindIII restriction site (underlined).The reverse primer (5Ј-CATGCGGATCCTCATTTGTGGAGGTA-GGCATCC-3Ј) contained the translation stop codon (italics) and a BamHI site (underlined).The PCR products were treated with HindIII and BamHI before cloning into p3ϫFlag.The plasmids are referred to as p3ϫFlag/⌬6 and p3ϫFlag/⌬6-53 HPGG 56 and allow the expression of N-terminally Flag-tagged desaturases.The integrity of the constructs and the presence of the desired deletions or mutations were assessed by DNA sequencing.