62
Phospholipid Synthesis by Chick Retinal Microsomes. Fatty Acid Preference and Effect of Fatty Acid Binding Protein Peggy A. Sellnera, b,* and Arlana R. Phllllpsa Departments of aAnatomy and Cell Biology and bOphthalmology, University of Kansas Medical Center, Kansas City, Kansas 66103
The acylation of 1-palmitoyl-sn-glycerophosphocholine (1-16:0-GPC) or 1-palmitoyl-sn-glycerophosphoethanol. amine (1-16:{~GPE) was measured using the microsomal fraction prepared from retinas of 14-15-day~ld chick embryos. Rates of incorporation of exogenously supplied fatty acids into diacyl-GPC were generally 5-7 times greater than into diacyl-GPE. Substrate preferences for incorporation into diacyl-GPC and diacyl-GPE were, respectively, 18:2 > 18:3 = 20:5 > 20:4 > 18:1 > 22:6 = 18:0 and 18:2 > 22:6 >t 18:3 = 18:0 t> 20:4 = 18:1 > 20:5. The apparent selectivities were not consistent with the reported fatty acid compositions of these lipid classes. The addition of partially purified fatty acid binding protein (FABP) to the reaction had no effect either on overall rates of incorporation or on the substrate preference. When fatty acyl-CoA substrates were used, rates of incorporation of the 18:0 derivative were much higher than with the fatty acid, while rates with other fatty acyl-CoA were similar to those with the respective fatty acid. Substrate preferences for CoA derivatives incorporated into diacyl-GPC were: 18:0 > 20:4 > 18:2 >I22:6, and into diacylGPE: 20:4 = 22:6 > 18:0 > 18:2. Polyunsaturated fatty acyl CoA (PUFA-CoA) were thus favored for incorporation into diacyl-GPE, and to a lesser extent into diacylGPC, a result that is consistent with composition data. When purified FABP was added to the reactions, there was an increase in the incorporation of 18:0-COA and a decrease or no change in the incorporation of PUFA-CoA. The deacylation/reacylation cycle thus appears to play a role in the modification of phospholipid composition. The data are not consistent, however, with a role for FABP in directing PUFA toward membrane lipid synthesis. Lipids 26, 62-67 (1991).
The fatty acid composition of retinas, like other neural tissue, is remarkably high in polyunsaturated fatty acids {PUFA), particularly of the n-3 family {1,2). Within the retina, the concentration of n-3 fatty acids is highest in the photoreceptor outer segments, where 22:6n-3 alone may constitute over 50% of fatty acids present in a given phospholipid class (1,3). Within the major phospholipid classes, P U F A are more prevalent among diacyl-GPE than among diacyl-GPC, For example, in adult chicken retina, P U F A constitute 48.5% of fatty acids in diacylGPE, but only 4.5% in diacyl-GPC (4). The incorporation of PUFA into phospholipids first requires the activation of the fatty acid to its coenzyme A *To whom correspondence should be addressed at the Department of Anatomy and CellBiology,Universityof Kansas MedicalCenter, 39th and Rainbow Boulevard, Kansas City, KS 66103. Abbreviations: ATP, adenosine triphosphate; CoA, Coenzyme A; DTT, dithiothreitol; FABP, fatty acid binding protein; GPC, glycerophosphocholine; GPE, glycerophosphoethanol8mine; GP, glycerophosphate; PMSF, phenylmethylsulfonyl fluoride; PUFA, polyunsaturated fatty acids; ROS, rod outer segments; RPE, retinal pigment epithelium; fatty acids are listed as chain length:number of double bonds. LIPIDS,Vol, 26, No. 1 (1991)
{CoA) derivative via the enzyme long-chain acyl-CoA synthetase. T w o forms of the enzyme are generally recognized, one that is non-specific and one that is selective for arachidonic acid. The non-specific form is present in microsomes, mitochondria, and peroxisomes of liver (5,6). The cDNA for this protein has recently been shown to be expressed in liver and heart, and to a much lesser extent in brain (7). The arachidonic acid-specific form of acylCoA synthetase has been described in blood vessel endothelium (8), brain (9) and retina (10). In the latter two tissues, activation of docosahexaenoic acid was also demonstrated with a Km similar to that for arachidonic acid (11,12). Thus, this 20:4-selective CoA synthetase may play an important role in the incorporation of these two P U F A into phospholipids. The fatty acyl-CoA can then serve as a substrate for acyl-CoA:l-acyl-GP acyltransferase for de novo synthesis of phosphatidic acid, or for acyl-CoA:l-acyl-GPC {or -GPE) acyltransferaee, involved in the tailoring of existing diacyl-GPC (or -GPE). The specificity of these acyltransferase enzymes for particular fatty acids could influence the composition of a particular membrane. For example, retinal microsomes can incorporate [3H]22:6 into phosphatidic acid (13,14) suggesting the introduction of long-chain P U F A during de n o v o synthesis. In other tissues the deacylation]reacylation cycle is involved in the restructuring of de n o v o synthesized phospholipids {15). Lands et aL (16) have shown that the reacylation of 1-acylGPC in rat liver preferentially incorporates PUFA. That this latter pathway also exists in the photoreceptor is consistent with the detection of phospholipase A2 activity in photoreceptor outer segments (17-19), thus providing a means of generating the lysophospholipids used in the acyltransferase reaction. Differences in turnover rates of individual molecular species of phospholipids in the retina (20) also imply active replacement of part or all of the membrane lipid molecules. The above reactions involve the intraceUular trafficking of fatty acids between cytoplasmic and/or membrane compartments; this movement may involve one or more binding proteins as intracellular carriers. Fatty acid binding protein (FABP) is a 12-14,000 dalton protein found in the cytosol of many tissues (for reviews, see refs. 21-24) including retina (25). This protein has been reported to have a binding preference for unsaturated fatty acids or their CoA esters (22), and has been suggested to play a role in directing these substrates to various metabolic pathways such as oxidation or esterification. For example, F A B P stimulates the activities of acyl-CoA:glycerol3-phosphate acyltransferase (26-28) and acetyl-CoA carboxylaee (29). Peeters et aL (30) have demonstrated the ability of liver and heart FABP to mediate the accelerated transfer of fatty acids between mitochondria and lipid vesicles. While functional studies with retinal FABP have not been performed, this protein could be an important mechanism for channelling PUFA into the phospholipids of outer segment membranes. We investigated the reacylation reaction in embryonic
63 FATTY ACID INCORPORATION INTO RETINA PHOSPHOLIPIDS chick retinas using synthetic lysophospholipids and seven fatty acids as the substrates. 1-Acyl-GPC and 1-acyl-GPE acyltransferase activities were measured using the microsomal fraction prepared from these retinas. F A B P was partially purified from the retina cytosol, and its effect on the reaction was measured. Under these conditions, rates of incorporation of fatty acid did not correlate well with composition data, and F A B P did not significantly influence either the reaction rate or the inherent substrate preference of the reactions. However, when CoA esters of four fatty acids were prepared and tested, there was greater incorporation of PUFA into diacyl-GPE and, to a lesser extent, into diacyl-GPC. The addition of purified F A B P to this reaction increased the incorporation of the saturated fatty acid and decreased the incorporation of PUFA. MATERIALS AND METHODS
Fertilized chicken eggs were obtained from a local supplier and were incubated for 14-15 days. Embryos were sacrificed by decapitation. The anterior segment of the eye was cut away and the vitreous was lifted out. A few drops of 0.1 M Tris-HC1, pH 7.8 {containing the following protease inhibitors in ~g/mL: phenylmethylsulfonyl fluoride (PMSF), 2; pepstatin, 3.4; aprotinin, 5; leupeptin, 2.4; and chymostatin, 5) were added to facilitate peeling away of the retina with fine forceps. At the region of the pecten, the retina was loosened by stroking with the tip of the forceps. At this stage of embryonic develol~ ment, the retinal pigment epithelium does not adhere to the retina, which can thus be isolated cleanly. Retinas were kept on ice in a small volume of Tris-HC1 plus protease inhibitors and were homogenized in a glass Potter-Elvehjem homogenizer. The homogenate was centrifuged at 12,000 X g for 20 rain to remove cell debris. The supernatant was then centrifuged at 100,000 X g to pellet the microsomal fraction, which was resuspended in 0.1 M Tris-HC1, pH 7.8 plus 5 mM dithiothreitol (DTT). The high-speed supernatant was layered onto a Sephadex G-100 column and eluted with 0.1 M Tris-HC1 buffer, pH 7.8, containing 0.2% sodium azide. The elution volume of FABP was determined in initial preparations by mixing an aliquot of supernatant with a trace amount of [1-14C]oleic acid and counting 200 ~L from each of the eluted fractions. In subsequent preparations, non-labelled fractions containing F A B P were pooled and concentrated using an Amicon ultrafiltration cell. This concentrated " F A B P fraction" was used in most of the enzyme assays. F A B P was not purified further for these experiments; this allowed for the use of microsomes and F A B P from the same preparation. All of the above procedures were carried out at 0-4~ In the experiments with fatty acyl-CoA derivatives, F A B P was further purified {Sellner, P.A., and Phillips, A.R., manuscript in preparation}. Briefly, the " F A B P fraction" prepared as described above was separated by native polyacrylamide gel electrophoresis. After staining one of the lanes with Coomassie Blue to locate the FABP band, the corresponding regions from the remaining gel were cut. F A B P was then eluted from the gel using the BioRad Electroeluter and concentrated by lyopbillzation. In most experiments, all embryos {7 dozen} were sacrificed on the same day, and microsomes were kept
refrigerated overnight to be used the following day after the F A B P fraction had been prepared. Acyltransferase activity was lost or greatly compromised if the microsomes were stored at - 2 0 ~ or if they were kept refrigerated for 2 days. Preparation of fatty acyl-CoA. Derivatives of [1-14C]18:0, 18:2, 20:4, and 22:6 were prepared according to the method of Schmidt and Burns {31}. We used 200 nmol of unlabelled fatty acid with 2 ~Ci labelled fatty acid in the synthesis to achieve a final concentration in our assays of 100 ~M fatty acyl-CoA. Acyltransferase assays. Each assay contained the following in a total volume of 0.1 mL: 0.1 mM 1-16:0-GPC or -GPE {pipetted in chloroform/methanol solution onto the bottom of the vial, solvent was evaporated with a stream of nitrogen}, 0.1 M Tris-HC1, pH 7.8 plus 5 mM dithiothreitol (DTT), I mM adenosine triphosphate (ATP), 10 mM MgC12, 0.1 mM CoA, and 100 tnM (0.1 /~Ci) [1-14C]fatty acid {18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3, or 22:6n-3), added in 5/~L of propylene glycol. The fatty acyl-CoA {18:0, 18:2, 20:4, or 22:6) was added in 40 ~L of aqueous suspension. The solution was vigorously vortexed after the addition of each constituent. Following a 5-min preincubation, 0.1-0.2 mg microsomal protein in 10 ~L was added, and the reaction was allowed to proceed at 37~ for 6 min. For those assays containing FABP fraction, the protein was added {10-20 ~g in 10 p_L} after the preincubation. Another 5-min incubation was allowed prior to the addition of microsomes. The reaction was stopped by the addition of 0.9 mL of CHCIJMeOH (2:1, v/v). Lipids were extracted by the method of Folch et al. (32}, and separated by thin-layer chromatography on 0.25 mm Silica Gel H plates (Analtech, Newark, DE) containing 7.5% magnesium acetate. The plate was developed twice in CHC1JMeOH/H20 (60:30:5, v/v/v), each time one-third the way up; then in hexane]diethyl ether/ formic acid (80:20:2, v/v/v), developed to within 1 cm from the top {33}. Standard lanes were sprayed with 2',7'-dichlorofluorescein and spots were visualized under ultraviolet light. The phospholipid and free fatty acid spots were scraped directly into scintillation vials and counted. Counts were converted to nmol after determining the specific activity from a 1 I~L aliquot of the fatty acidpropylene glycol mixture. The data are expressed as umol diacyl-GPC or -GPE formed/mg protein/6 min. Protein was determined by the method of Lowry et al. 134). Non-radiolabelled fatty acids were obtained from Sigma Chemical Company (St. Louis, MO); radiolabelled fatty acids were from Dupont/New England Nuclear {Wilmington, DE}. All other chemicals were of reagent grade. RESULTS The elution profile of retina cytosolic fraction (high-speed supernatant} mixed with [1-14C]18:1 from a Sephadex G-100 column is shown in Figure 1. FABP elutes between 130-160 mL; the peak corresponds to a molecular weight of around 12,000 daltons. When an aliquot of this fraction with a trace amount of [1-14C]18:1 methyl ester is electrophoresed on a native polyacrylamide gel, only one band {FABP) contains radioactivity, though several bands are present upon staining with Coomassie Blue {data not shown}. Thus, we reasoned that any effect of LIPIDS, Vol. 26, No, 1 (1991)
64 P.A. SELLNER AND A.R. PHILLIPS TABLE 2
~
Sephadex G-IO0
0.8
Retina cytos~ + 14
0.7
C-18:1
Incorporation of Fatty Acids into Diacyl-GPE and the Effect of F A B P a
3000
nmol GPE formed]rag proteird6 rain
2500
0.6
Fatty acid 2000
0.5
o
1500
T 1000
02
,';"'"",-~ 500
"t Y 50
100
"
1SO 200 Elutlon v~um~ ml
250
FIG. 1. Elution of F A B P fraction in retina cytosol. An aliquot of retina cytosolic fraction was mixed with a trace amount (0.5 ~Ci) of [14C]18:1. The labelled cytosol was e h t e d from a column of Sephadex G-100 as described in Materials and Methods. The solid line represents protein {OD2~; the dashed line represents fatty add bound to protein (cpm). In subsequent runs without added fatty add, the fractions marked "FABP" were pooled, concentrated, and used without further purification.
TABLE 1 Incorporation of Fatty Acids into Diacyl-GPC and the Effect of FABP a
nmol GPC formed/mg protein/6 min no FABP + FABP
Fatty acid 18:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3
0.44 1.18 3.24 2.50 1.58 2.53 0.37
+__0.12 +__0.58 + 0.94 +__0.84 ___0.64 + 0.69 +_ 0.19
(4) {5) (5) (5) {4) (5} {5)
0.46 1.11 3.66 1.92 1.04 2.69 0.44
--4---0.25 (4) -!-_0.51 (5) +_ 1.20 (4) --4-_0.80 (5) + 0.72 {4) +_ 1.03 (4) +_ 0.32 (4)
aAcyltransferase activities were measured as described in Materials and Methods, using 1-palmitoyl-sn-GPC and the indicated [14C]labelled fatty acid as substrates. Where indicated, FABP fraction (20 ~g total protein} was added prior to addition of microsomes. Values represent means _ SEM for the number of experiments indicated in parentheses
the addition of this fraction on the reaction was attributable to F A B P . The incorporation of f a t t y acid into diacyl-GPC and the effect of F A B P is shown in Table 1. While the absolute reaction r a t e s varied considerably between experiments, within a given experiment, some general trends could be observed. For 1-16:0-GPC, the m a x i m u m r a t e s of incorporation were with 18:2 as the substrate, followed b y 18:3 = 20:5 > 20:4 > 18:1 > 22:6 = 18:0. The incorporation of 18:0 was low, as would be expected for acylation of the 2-position of the phospholipid, b u t r a t e s for the incorporation of 20:4 and 22:6 were also lower t h a n would be expected f r o m the composition d a t a (4). LIPIDS, Vol. 26, No. 1 (1991)
18:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:611-3
no FABP 0.34 0.24 0.60 0.36 0.27 0.17 0.40
+ 0.18 (4} + 0.10 (5) -+ 0.23 (5} + 0.13 (3) + 0.16 (3) + 0.05 (5) + 0.15 (3)
+ FABP 0.28 0.26 0.68 0.92 0.26 0.26 0.20
+ 0.11 (4) + 0.07 (4) _ 0.39 (3) +_ 0.56 (4) +_ 0.14 (4) + 0.04 (3) +_ 0.03 (2)
aAcyltransferase activities were measured as described in Materials and Methods, using 1-palmitoyl-sn-GPE and the indicated [14C]labelled fatty acids as substrates. Other information is as for Table 1.
The addition of 20 ~g of F A B P fraction caused a decrease (not statistically significant) in the incorporation of 18:1, 18:3, and 20:4, and a slight increase in r a t e s with 18:0, 18:2, 20:5, and 22:6. In conjunction with these minor effects on absolute reaction rates, the effect of F A B P on the overall s u b s t r a t e preference was minimal. Thus, for diacyl-GPC, 18:2 was again the s u b s t r a t e with maximal rates of incorporation, followed b y 20:5 > 18:3 > 18:1 = 20:4 > 18:0 = 22:6. This represents a trend toward increasing the incorporation of 20:5 over 18:3 and decreasing the incorporation of 20:4 relative to 18:1 in the absence of F A B P . R a t e s of incorporation of f a t t y acids into diacyl-GPE and the effects of F A B P are given in Table 2. Again, 18:2 was the s u b s t r a t e with the highest rates of incorporation, followed b y 22:6 >t 18:3 = 18:0/> 20:4 = 18:1 > 20:5. In the presence of F A B P , the incorporation of 18:3 was increased substantially; m o s t of this increase is due to the result of one e x p e r i m e n t in which an unusually large a m o u n t of this f a t t y acid was incorporated. Smaller increases were seen with 18:1, 18:2, and 20:5, while the reaction rates with 18:0, 20:4, and 22:6 declined. The substrate preference was changed to 18:3 > 18:2 > 18.'0 = 18:1 = 20:5 = 20:4 >I 22:6. There was a greater similarity of reaction r a t e s a m o n g m a n y of the f a t t y acids, and a trend, surprisingly, toward decreasing the incorporation of 22:6 into this phospholipid. Incorporation r a t e s with 1-16:0-GPE were generally 5-7-fold lower t h a n for 1-16:0-GPC. I n t e r e s t i n g exceptions were the r a t e s of 18:0 and 22:6 incorporation, which were nearly equal in the two phospholipids, and 20:5, which had r a t e s of incorporation into diacyl-GPC t h a t were 15-fold g r e a t e r t h a n into diacyl-GPE. I n neither phospholipid was the p a t t e r n of s u b s t r a t e preference consistent with the composition of P U F A in these membrane lipids. To b y p a s s the CoA activation step and t h u s eliminate a n y selectivity the acyl-CoA s y n t h e t a s e m i g h t have on the incorporation, we performed an experiment using synthesized f a t t y acyl-CoA derivatives {Table 3). Rates were generally similar to those obtained with the f a t t y acids, except for a 10-fold higher r a t e of incorporation of 18:0-CoA into diacyl-GPC. There were some differences in s u b s t r a t e preference: for diacyl-GPC, 18:0 showed the
65 FATTY ACID INCORPORATION INTO RETINA PHOSPHOLIPIDS TABLE 3 Incorporation of Fatty Acyl-CoA into Diacyl~PC and Diacyl-GPE, and the Effect of FABP a
nmol formed]mg protein/6 min diacyl-GPC diacyl-GPE Fatty acyl-CoA no FABP + FABP no FABP + FABP 18:0 18:2n-6 20:4n-6 22:6n-3
5.39 0.58 1.87 0.31
6.04 0.17 1.46 0.23
1.65 0.15 2.32 2.19
3.21 0.22 1.04
1.38
aCoA esters of [14C]labelledfatty acids were prepared as described in Materials and Methods. Acyltransferase activities were measured as for Tables 1 and 2, except that 10 ~g of purified FABP was added rather than FABP fraction. Values represent the means of duplicate determinations.
highest rates of incorporation, followed by 20:4, 18:2, and 22:6. For diacyl-GPE, 20:4 and 22:6 were equally effective, followed by 18:0 and 18:2. Interestingly, the effect of F A B P was to increase the incorporation of 18:0, and to decrease or have no effect on the incorporation of the unsaturated fatty acids. DISCUSSION
The purpose of the high n-3 fatty acid content of retina is unclear, but is not limited to providing a highly fluid environment for the visual pigment (35,36). The function of n-3 fatty acids may be related to other characteristics of the outer segment disk membrane, such as flexibility, permeability, or the high degree of membrane turnover in the photoreceptor outer segment. Clearly unique mechanisms exist within the photoreceptor or photoreceptorretinal pigment epithelium (RPE) complex to create and maintain the unusual composition. One possibility is that the acyltransferase could prefer PUFA, as has been shown in other systems. Additionally, FABP could direct certain P U F A toward incorporation into lysophospholipids and away from oxidation or other routes, thus tailoring the composition of de novo synthesized molecular species. In these studies, rates of incorporation of fatty acids into diacyl-GPC were much greater than into diacyl-GPE, despite the fact that these phospholipids are present in approximately the same molar concentrations in these membranes (4). Masuzawa et al. {37) also found that 1acyl-GPC was the preferred acceptor over 1-acyl-GPE for the CoA- and ATP-dependent acylation. Similar results in retina were found by investigators using different experimental protocols. For example, when rat eyes were injected intravitreally with radiolabelled 20:5n-3, more label was recovered in the diacyl-GPC than diacyl-GPE fraction at all time points (38). Bovine retinas incubated in vitro with 20:4n-6, 20:5n-3, or 22:6n-3 labelled diacylGPC to a greater extent in all subcellular fractions than diacyl-GPE (39). This may suggest that in vivo the principal species synthesized is diacyl-GPC, and diacyl-GPE is prepared from this via base exchange or other conversion,
Louie et aL have found 10-fold greater specific activities among 22:6~containing species of diacyl-GPC than diacylGPE in frog rod outer segments (ROS) prepared from eyes injected with [3H]glycerol. In addition, half-lives of all 22:6-containing species were longer in diacyl-GPE than in diacyl-GPC. Similarly, in vitro experiments with isolated bovine ROS incubated with [3H]22:6 yielded specific activities of diacyl-GPC that were 10 times greater than those of diacyl-GPE or diacyl-glycerophosphoserine (40). Specific activities of 22:6-22:6 species were higher than any other 22:6-containing species for all phospholipids studied. Thus, while more diacyl-GPC is initially formed, it may also be rapidly turning over, a process which could not be estimated from our short incubations. Low rates of incorporation of 18:0 would be expected for the 2-position of GPC and GPE, and our observed rates were therefore consistent with composition data. However, the rates of incorporation with 18:0-CoA were much higher. We have no explanation for this, even though disaturated species of GPC have been isolated (41). The greatest rates of incorporation of fatty acid in our studies were seen with 18:2, although when the CoA esters were used, 18:2 was the least utilized substrate. This suggests that in the assays with fatty acids, conditions were optimal for the activation and incorporation of 18:2 over other substrates. The acyltransferase, on the other hand, is not selective for this fatty acyl-CoA. Johnston and Hudson (4) report 0.1 mol% of 18:2 in whole retina diacyl-GPC and 7.8 mol% in diacyl-GPE. We have found very little (
Phospholipid Synthesis by Chick Retinal Microsomes. Fatty Acid Preference and Effect of Fatty Acid Binding Protein Peggy A. Sellnera, b,* and Arlana R. Phllllpsa Departments of aAnatomy and Cell Biology and bOphthalmology, University of Kansas Medical Center, Kansas City, Kansas 66103
The acylation of 1-palmitoyl-sn-glycerophosphocholine (1-16:0-GPC) or 1-palmitoyl-sn-glycerophosphoethanol. amine (1-16:{~GPE) was measured using the microsomal fraction prepared from retinas of 14-15-day~ld chick embryos. Rates of incorporation of exogenously supplied fatty acids into diacyl-GPC were generally 5-7 times greater than into diacyl-GPE. Substrate preferences for incorporation into diacyl-GPC and diacyl-GPE were, respectively, 18:2 > 18:3 = 20:5 > 20:4 > 18:1 > 22:6 = 18:0 and 18:2 > 22:6 >t 18:3 = 18:0 t> 20:4 = 18:1 > 20:5. The apparent selectivities were not consistent with the reported fatty acid compositions of these lipid classes. The addition of partially purified fatty acid binding protein (FABP) to the reaction had no effect either on overall rates of incorporation or on the substrate preference. When fatty acyl-CoA substrates were used, rates of incorporation of the 18:0 derivative were much higher than with the fatty acid, while rates with other fatty acyl-CoA were similar to those with the respective fatty acid. Substrate preferences for CoA derivatives incorporated into diacyl-GPC were: 18:0 > 20:4 > 18:2 >I22:6, and into diacylGPE: 20:4 = 22:6 > 18:0 > 18:2. Polyunsaturated fatty acyl CoA (PUFA-CoA) were thus favored for incorporation into diacyl-GPE, and to a lesser extent into diacylGPC, a result that is consistent with composition data. When purified FABP was added to the reactions, there was an increase in the incorporation of 18:0-COA and a decrease or no change in the incorporation of PUFA-CoA. The deacylation/reacylation cycle thus appears to play a role in the modification of phospholipid composition. The data are not consistent, however, with a role for FABP in directing PUFA toward membrane lipid synthesis. Lipids 26, 62-67 (1991).
The fatty acid composition of retinas, like other neural tissue, is remarkably high in polyunsaturated fatty acids {PUFA), particularly of the n-3 family {1,2). Within the retina, the concentration of n-3 fatty acids is highest in the photoreceptor outer segments, where 22:6n-3 alone may constitute over 50% of fatty acids present in a given phospholipid class (1,3). Within the major phospholipid classes, P U F A are more prevalent among diacyl-GPE than among diacyl-GPC, For example, in adult chicken retina, P U F A constitute 48.5% of fatty acids in diacylGPE, but only 4.5% in diacyl-GPC (4). The incorporation of PUFA into phospholipids first requires the activation of the fatty acid to its coenzyme A *To whom correspondence should be addressed at the Department of Anatomy and CellBiology,Universityof Kansas MedicalCenter, 39th and Rainbow Boulevard, Kansas City, KS 66103. Abbreviations: ATP, adenosine triphosphate; CoA, Coenzyme A; DTT, dithiothreitol; FABP, fatty acid binding protein; GPC, glycerophosphocholine; GPE, glycerophosphoethanol8mine; GP, glycerophosphate; PMSF, phenylmethylsulfonyl fluoride; PUFA, polyunsaturated fatty acids; ROS, rod outer segments; RPE, retinal pigment epithelium; fatty acids are listed as chain length:number of double bonds. LIPIDS,Vol, 26, No. 1 (1991)
{CoA) derivative via the enzyme long-chain acyl-CoA synthetase. T w o forms of the enzyme are generally recognized, one that is non-specific and one that is selective for arachidonic acid. The non-specific form is present in microsomes, mitochondria, and peroxisomes of liver (5,6). The cDNA for this protein has recently been shown to be expressed in liver and heart, and to a much lesser extent in brain (7). The arachidonic acid-specific form of acylCoA synthetase has been described in blood vessel endothelium (8), brain (9) and retina (10). In the latter two tissues, activation of docosahexaenoic acid was also demonstrated with a Km similar to that for arachidonic acid (11,12). Thus, this 20:4-selective CoA synthetase may play an important role in the incorporation of these two P U F A into phospholipids. The fatty acyl-CoA can then serve as a substrate for acyl-CoA:l-acyl-GP acyltransferase for de novo synthesis of phosphatidic acid, or for acyl-CoA:l-acyl-GPC {or -GPE) acyltransferaee, involved in the tailoring of existing diacyl-GPC (or -GPE). The specificity of these acyltransferase enzymes for particular fatty acids could influence the composition of a particular membrane. For example, retinal microsomes can incorporate [3H]22:6 into phosphatidic acid (13,14) suggesting the introduction of long-chain P U F A during de n o v o synthesis. In other tissues the deacylation]reacylation cycle is involved in the restructuring of de n o v o synthesized phospholipids {15). Lands et aL (16) have shown that the reacylation of 1-acylGPC in rat liver preferentially incorporates PUFA. That this latter pathway also exists in the photoreceptor is consistent with the detection of phospholipase A2 activity in photoreceptor outer segments (17-19), thus providing a means of generating the lysophospholipids used in the acyltransferase reaction. Differences in turnover rates of individual molecular species of phospholipids in the retina (20) also imply active replacement of part or all of the membrane lipid molecules. The above reactions involve the intraceUular trafficking of fatty acids between cytoplasmic and/or membrane compartments; this movement may involve one or more binding proteins as intracellular carriers. Fatty acid binding protein (FABP) is a 12-14,000 dalton protein found in the cytosol of many tissues (for reviews, see refs. 21-24) including retina (25). This protein has been reported to have a binding preference for unsaturated fatty acids or their CoA esters (22), and has been suggested to play a role in directing these substrates to various metabolic pathways such as oxidation or esterification. For example, F A B P stimulates the activities of acyl-CoA:glycerol3-phosphate acyltransferase (26-28) and acetyl-CoA carboxylaee (29). Peeters et aL (30) have demonstrated the ability of liver and heart FABP to mediate the accelerated transfer of fatty acids between mitochondria and lipid vesicles. While functional studies with retinal FABP have not been performed, this protein could be an important mechanism for channelling PUFA into the phospholipids of outer segment membranes. We investigated the reacylation reaction in embryonic
63 FATTY ACID INCORPORATION INTO RETINA PHOSPHOLIPIDS chick retinas using synthetic lysophospholipids and seven fatty acids as the substrates. 1-Acyl-GPC and 1-acyl-GPE acyltransferase activities were measured using the microsomal fraction prepared from these retinas. F A B P was partially purified from the retina cytosol, and its effect on the reaction was measured. Under these conditions, rates of incorporation of fatty acid did not correlate well with composition data, and F A B P did not significantly influence either the reaction rate or the inherent substrate preference of the reactions. However, when CoA esters of four fatty acids were prepared and tested, there was greater incorporation of PUFA into diacyl-GPE and, to a lesser extent, into diacyl-GPC. The addition of purified F A B P to this reaction increased the incorporation of the saturated fatty acid and decreased the incorporation of PUFA. MATERIALS AND METHODS
Fertilized chicken eggs were obtained from a local supplier and were incubated for 14-15 days. Embryos were sacrificed by decapitation. The anterior segment of the eye was cut away and the vitreous was lifted out. A few drops of 0.1 M Tris-HC1, pH 7.8 {containing the following protease inhibitors in ~g/mL: phenylmethylsulfonyl fluoride (PMSF), 2; pepstatin, 3.4; aprotinin, 5; leupeptin, 2.4; and chymostatin, 5) were added to facilitate peeling away of the retina with fine forceps. At the region of the pecten, the retina was loosened by stroking with the tip of the forceps. At this stage of embryonic develol~ ment, the retinal pigment epithelium does not adhere to the retina, which can thus be isolated cleanly. Retinas were kept on ice in a small volume of Tris-HC1 plus protease inhibitors and were homogenized in a glass Potter-Elvehjem homogenizer. The homogenate was centrifuged at 12,000 X g for 20 rain to remove cell debris. The supernatant was then centrifuged at 100,000 X g to pellet the microsomal fraction, which was resuspended in 0.1 M Tris-HC1, pH 7.8 plus 5 mM dithiothreitol (DTT). The high-speed supernatant was layered onto a Sephadex G-100 column and eluted with 0.1 M Tris-HC1 buffer, pH 7.8, containing 0.2% sodium azide. The elution volume of FABP was determined in initial preparations by mixing an aliquot of supernatant with a trace amount of [1-14C]oleic acid and counting 200 ~L from each of the eluted fractions. In subsequent preparations, non-labelled fractions containing F A B P were pooled and concentrated using an Amicon ultrafiltration cell. This concentrated " F A B P fraction" was used in most of the enzyme assays. F A B P was not purified further for these experiments; this allowed for the use of microsomes and F A B P from the same preparation. All of the above procedures were carried out at 0-4~ In the experiments with fatty acyl-CoA derivatives, F A B P was further purified {Sellner, P.A., and Phillips, A.R., manuscript in preparation}. Briefly, the " F A B P fraction" prepared as described above was separated by native polyacrylamide gel electrophoresis. After staining one of the lanes with Coomassie Blue to locate the FABP band, the corresponding regions from the remaining gel were cut. F A B P was then eluted from the gel using the BioRad Electroeluter and concentrated by lyopbillzation. In most experiments, all embryos {7 dozen} were sacrificed on the same day, and microsomes were kept
refrigerated overnight to be used the following day after the F A B P fraction had been prepared. Acyltransferase activity was lost or greatly compromised if the microsomes were stored at - 2 0 ~ or if they were kept refrigerated for 2 days. Preparation of fatty acyl-CoA. Derivatives of [1-14C]18:0, 18:2, 20:4, and 22:6 were prepared according to the method of Schmidt and Burns {31}. We used 200 nmol of unlabelled fatty acid with 2 ~Ci labelled fatty acid in the synthesis to achieve a final concentration in our assays of 100 ~M fatty acyl-CoA. Acyltransferase assays. Each assay contained the following in a total volume of 0.1 mL: 0.1 mM 1-16:0-GPC or -GPE {pipetted in chloroform/methanol solution onto the bottom of the vial, solvent was evaporated with a stream of nitrogen}, 0.1 M Tris-HC1, pH 7.8 plus 5 mM dithiothreitol (DTT), I mM adenosine triphosphate (ATP), 10 mM MgC12, 0.1 mM CoA, and 100 tnM (0.1 /~Ci) [1-14C]fatty acid {18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3, or 22:6n-3), added in 5/~L of propylene glycol. The fatty acyl-CoA {18:0, 18:2, 20:4, or 22:6) was added in 40 ~L of aqueous suspension. The solution was vigorously vortexed after the addition of each constituent. Following a 5-min preincubation, 0.1-0.2 mg microsomal protein in 10 ~L was added, and the reaction was allowed to proceed at 37~ for 6 min. For those assays containing FABP fraction, the protein was added {10-20 ~g in 10 p_L} after the preincubation. Another 5-min incubation was allowed prior to the addition of microsomes. The reaction was stopped by the addition of 0.9 mL of CHCIJMeOH (2:1, v/v). Lipids were extracted by the method of Folch et al. (32}, and separated by thin-layer chromatography on 0.25 mm Silica Gel H plates (Analtech, Newark, DE) containing 7.5% magnesium acetate. The plate was developed twice in CHC1JMeOH/H20 (60:30:5, v/v/v), each time one-third the way up; then in hexane]diethyl ether/ formic acid (80:20:2, v/v/v), developed to within 1 cm from the top {33}. Standard lanes were sprayed with 2',7'-dichlorofluorescein and spots were visualized under ultraviolet light. The phospholipid and free fatty acid spots were scraped directly into scintillation vials and counted. Counts were converted to nmol after determining the specific activity from a 1 I~L aliquot of the fatty acidpropylene glycol mixture. The data are expressed as umol diacyl-GPC or -GPE formed/mg protein/6 min. Protein was determined by the method of Lowry et al. 134). Non-radiolabelled fatty acids were obtained from Sigma Chemical Company (St. Louis, MO); radiolabelled fatty acids were from Dupont/New England Nuclear {Wilmington, DE}. All other chemicals were of reagent grade. RESULTS The elution profile of retina cytosolic fraction (high-speed supernatant} mixed with [1-14C]18:1 from a Sephadex G-100 column is shown in Figure 1. FABP elutes between 130-160 mL; the peak corresponds to a molecular weight of around 12,000 daltons. When an aliquot of this fraction with a trace amount of [1-14C]18:1 methyl ester is electrophoresed on a native polyacrylamide gel, only one band {FABP) contains radioactivity, though several bands are present upon staining with Coomassie Blue {data not shown}. Thus, we reasoned that any effect of LIPIDS, Vol. 26, No, 1 (1991)
64 P.A. SELLNER AND A.R. PHILLIPS TABLE 2
~
Sephadex G-IO0
0.8
Retina cytos~ + 14
0.7
C-18:1
Incorporation of Fatty Acids into Diacyl-GPE and the Effect of F A B P a
3000
nmol GPE formed]rag proteird6 rain
2500
0.6
Fatty acid 2000
0.5
o
1500
T 1000
02
,';"'"",-~ 500
"t Y 50
100
"
1SO 200 Elutlon v~um~ ml
250
FIG. 1. Elution of F A B P fraction in retina cytosol. An aliquot of retina cytosolic fraction was mixed with a trace amount (0.5 ~Ci) of [14C]18:1. The labelled cytosol was e h t e d from a column of Sephadex G-100 as described in Materials and Methods. The solid line represents protein {OD2~; the dashed line represents fatty add bound to protein (cpm). In subsequent runs without added fatty add, the fractions marked "FABP" were pooled, concentrated, and used without further purification.
TABLE 1 Incorporation of Fatty Acids into Diacyl-GPC and the Effect of FABP a
nmol GPC formed/mg protein/6 min no FABP + FABP
Fatty acid 18:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3
0.44 1.18 3.24 2.50 1.58 2.53 0.37
+__0.12 +__0.58 + 0.94 +__0.84 ___0.64 + 0.69 +_ 0.19
(4) {5) (5) (5) {4) (5} {5)
0.46 1.11 3.66 1.92 1.04 2.69 0.44
--4---0.25 (4) -!-_0.51 (5) +_ 1.20 (4) --4-_0.80 (5) + 0.72 {4) +_ 1.03 (4) +_ 0.32 (4)
aAcyltransferase activities were measured as described in Materials and Methods, using 1-palmitoyl-sn-GPC and the indicated [14C]labelled fatty acid as substrates. Where indicated, FABP fraction (20 ~g total protein} was added prior to addition of microsomes. Values represent means _ SEM for the number of experiments indicated in parentheses
the addition of this fraction on the reaction was attributable to F A B P . The incorporation of f a t t y acid into diacyl-GPC and the effect of F A B P is shown in Table 1. While the absolute reaction r a t e s varied considerably between experiments, within a given experiment, some general trends could be observed. For 1-16:0-GPC, the m a x i m u m r a t e s of incorporation were with 18:2 as the substrate, followed b y 18:3 = 20:5 > 20:4 > 18:1 > 22:6 = 18:0. The incorporation of 18:0 was low, as would be expected for acylation of the 2-position of the phospholipid, b u t r a t e s for the incorporation of 20:4 and 22:6 were also lower t h a n would be expected f r o m the composition d a t a (4). LIPIDS, Vol. 26, No. 1 (1991)
18:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:611-3
no FABP 0.34 0.24 0.60 0.36 0.27 0.17 0.40
+ 0.18 (4} + 0.10 (5) -+ 0.23 (5} + 0.13 (3) + 0.16 (3) + 0.05 (5) + 0.15 (3)
+ FABP 0.28 0.26 0.68 0.92 0.26 0.26 0.20
+ 0.11 (4) + 0.07 (4) _ 0.39 (3) +_ 0.56 (4) +_ 0.14 (4) + 0.04 (3) +_ 0.03 (2)
aAcyltransferase activities were measured as described in Materials and Methods, using 1-palmitoyl-sn-GPE and the indicated [14C]labelled fatty acids as substrates. Other information is as for Table 1.
The addition of 20 ~g of F A B P fraction caused a decrease (not statistically significant) in the incorporation of 18:1, 18:3, and 20:4, and a slight increase in r a t e s with 18:0, 18:2, 20:5, and 22:6. In conjunction with these minor effects on absolute reaction rates, the effect of F A B P on the overall s u b s t r a t e preference was minimal. Thus, for diacyl-GPC, 18:2 was again the s u b s t r a t e with maximal rates of incorporation, followed b y 20:5 > 18:3 > 18:1 = 20:4 > 18:0 = 22:6. This represents a trend toward increasing the incorporation of 20:5 over 18:3 and decreasing the incorporation of 20:4 relative to 18:1 in the absence of F A B P . R a t e s of incorporation of f a t t y acids into diacyl-GPE and the effects of F A B P are given in Table 2. Again, 18:2 was the s u b s t r a t e with the highest rates of incorporation, followed b y 22:6 >t 18:3 = 18:0/> 20:4 = 18:1 > 20:5. In the presence of F A B P , the incorporation of 18:3 was increased substantially; m o s t of this increase is due to the result of one e x p e r i m e n t in which an unusually large a m o u n t of this f a t t y acid was incorporated. Smaller increases were seen with 18:1, 18:2, and 20:5, while the reaction rates with 18:0, 20:4, and 22:6 declined. The substrate preference was changed to 18:3 > 18:2 > 18.'0 = 18:1 = 20:5 = 20:4 >I 22:6. There was a greater similarity of reaction r a t e s a m o n g m a n y of the f a t t y acids, and a trend, surprisingly, toward decreasing the incorporation of 22:6 into this phospholipid. Incorporation r a t e s with 1-16:0-GPE were generally 5-7-fold lower t h a n for 1-16:0-GPC. I n t e r e s t i n g exceptions were the r a t e s of 18:0 and 22:6 incorporation, which were nearly equal in the two phospholipids, and 20:5, which had r a t e s of incorporation into diacyl-GPC t h a t were 15-fold g r e a t e r t h a n into diacyl-GPE. I n neither phospholipid was the p a t t e r n of s u b s t r a t e preference consistent with the composition of P U F A in these membrane lipids. To b y p a s s the CoA activation step and t h u s eliminate a n y selectivity the acyl-CoA s y n t h e t a s e m i g h t have on the incorporation, we performed an experiment using synthesized f a t t y acyl-CoA derivatives {Table 3). Rates were generally similar to those obtained with the f a t t y acids, except for a 10-fold higher r a t e of incorporation of 18:0-CoA into diacyl-GPC. There were some differences in s u b s t r a t e preference: for diacyl-GPC, 18:0 showed the
65 FATTY ACID INCORPORATION INTO RETINA PHOSPHOLIPIDS TABLE 3 Incorporation of Fatty Acyl-CoA into Diacyl~PC and Diacyl-GPE, and the Effect of FABP a
nmol formed]mg protein/6 min diacyl-GPC diacyl-GPE Fatty acyl-CoA no FABP + FABP no FABP + FABP 18:0 18:2n-6 20:4n-6 22:6n-3
5.39 0.58 1.87 0.31
6.04 0.17 1.46 0.23
1.65 0.15 2.32 2.19
3.21 0.22 1.04
1.38
aCoA esters of [14C]labelledfatty acids were prepared as described in Materials and Methods. Acyltransferase activities were measured as for Tables 1 and 2, except that 10 ~g of purified FABP was added rather than FABP fraction. Values represent the means of duplicate determinations.
highest rates of incorporation, followed by 20:4, 18:2, and 22:6. For diacyl-GPE, 20:4 and 22:6 were equally effective, followed by 18:0 and 18:2. Interestingly, the effect of F A B P was to increase the incorporation of 18:0, and to decrease or have no effect on the incorporation of the unsaturated fatty acids. DISCUSSION
The purpose of the high n-3 fatty acid content of retina is unclear, but is not limited to providing a highly fluid environment for the visual pigment (35,36). The function of n-3 fatty acids may be related to other characteristics of the outer segment disk membrane, such as flexibility, permeability, or the high degree of membrane turnover in the photoreceptor outer segment. Clearly unique mechanisms exist within the photoreceptor or photoreceptorretinal pigment epithelium (RPE) complex to create and maintain the unusual composition. One possibility is that the acyltransferase could prefer PUFA, as has been shown in other systems. Additionally, FABP could direct certain P U F A toward incorporation into lysophospholipids and away from oxidation or other routes, thus tailoring the composition of de novo synthesized molecular species. In these studies, rates of incorporation of fatty acids into diacyl-GPC were much greater than into diacyl-GPE, despite the fact that these phospholipids are present in approximately the same molar concentrations in these membranes (4). Masuzawa et al. {37) also found that 1acyl-GPC was the preferred acceptor over 1-acyl-GPE for the CoA- and ATP-dependent acylation. Similar results in retina were found by investigators using different experimental protocols. For example, when rat eyes were injected intravitreally with radiolabelled 20:5n-3, more label was recovered in the diacyl-GPC than diacyl-GPE fraction at all time points (38). Bovine retinas incubated in vitro with 20:4n-6, 20:5n-3, or 22:6n-3 labelled diacylGPC to a greater extent in all subcellular fractions than diacyl-GPE (39). This may suggest that in vivo the principal species synthesized is diacyl-GPC, and diacyl-GPE is prepared from this via base exchange or other conversion,
Louie et aL have found 10-fold greater specific activities among 22:6~containing species of diacyl-GPC than diacylGPE in frog rod outer segments (ROS) prepared from eyes injected with [3H]glycerol. In addition, half-lives of all 22:6-containing species were longer in diacyl-GPE than in diacyl-GPC. Similarly, in vitro experiments with isolated bovine ROS incubated with [3H]22:6 yielded specific activities of diacyl-GPC that were 10 times greater than those of diacyl-GPE or diacyl-glycerophosphoserine (40). Specific activities of 22:6-22:6 species were higher than any other 22:6-containing species for all phospholipids studied. Thus, while more diacyl-GPC is initially formed, it may also be rapidly turning over, a process which could not be estimated from our short incubations. Low rates of incorporation of 18:0 would be expected for the 2-position of GPC and GPE, and our observed rates were therefore consistent with composition data. However, the rates of incorporation with 18:0-CoA were much higher. We have no explanation for this, even though disaturated species of GPC have been isolated (41). The greatest rates of incorporation of fatty acid in our studies were seen with 18:2, although when the CoA esters were used, 18:2 was the least utilized substrate. This suggests that in the assays with fatty acids, conditions were optimal for the activation and incorporation of 18:2 over other substrates. The acyltransferase, on the other hand, is not selective for this fatty acyl-CoA. Johnston and Hudson (4) report 0.1 mol% of 18:2 in whole retina diacyl-GPC and 7.8 mol% in diacyl-GPE. We have found very little (
