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The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes
Comparative Biochemistry and Physiology Part B 120 (1998) 785 – 789
The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes Ching Yuan a, Huiming Chen c,e,f, Robert E. Anderson c,d,e,f, Osamu Kuwata b, Thomas G. Ebrey a,b,* b
a Center for Biophysics and Computational Biology, Uni6ersity of Illinois at Urbana-Champaign, Urbana, IL, USA Department of Cell and Structural Biology, Uni6ersity of Illinois at Urbana-Champaign, 601 South Goodwin A6enue, Urbana, IL 61801, USA c Department of Ophthalmology, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA d Biochemistry & Molecular Biology, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA e Oklahoma Center for Neuroscience, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA f Dean A. McGee Eye Institute, Oklahoma City, OK, USA
Received 5 March 1998; received in revised form 22 June 1998; accepted 1 July 1998
Abstract This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Cell membrane; Lipid composition; Gecko; Retina; Photoreceptor; Cone-type visual pigment; Docosahexaenoic acid; Phospholipid
1. Introduction The composition and molecular species of the lipids in photoreceptor outer segment membranes have been investigated extensively for several types of vertebrates, mostly mammals and amphibians [1,2,8,19]. A remarkable similarity was observed for the various phospholipid species: PC, 40 – 50%; PE, 30 – 35%; PS, 5 –15%; phosphatidylinositol (PI), 3 – 6%; diacyl glyceride (DG), 1 – 2%; and phosphatidic acid (PA), less than 1%. The phospholipid composition is known to influence the kinetics and equilibrium of the Meta I/Meta II intermediates of rhodopsin [11,12] and the kinetics and extent of phosphodiesterase activation [4]. * Corresponding author. Tel.: +1 217 3332015; fax: + 1 217 2446615; e-mail: [email protected] 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10079-2
The retinas of the animal species studied so far have only a small percentage of their total visual pigments as cone pigments. It would be interesting to know whether the lipid composition of photoreceptor cells with conetype pigments is different from those with rod-type pigments and whether cone-type pigments require a distinct lipid environment for their proper function compared to rhodopsin. The gecko retina contains almost exclusively rod shaped photoreceptor cells. However, more than 90% of the visual pigment in these photoreceptor cells is a cone-type pigment, P521 [7]. In this report we determined the fatty acid and phospholipid class composition of gecko photoreceptor outer segment membranes. Our results show that its composition is somewhat different from what is found in photoreceptors containing rod-type pigments. In gecko
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photoreceptors, there is more negatively charged phospholipid, PS, compared to bovine and frog. Furthermore, a surprisingly high percentage of DHA is found in PC and PS isolated from gecko photoreceptor membranes. The molar percentage of these two phospholipid species are also higher in the gecko photoreceptor outer segment membranes.
2. Materials and methods
2.1. Preparation of gecko photoreceptor outer segments Tokay geckos from WestCoast Reptile (Fullerton, CA) were dark-adapted for at least 12 h at room temperature. The animals were then cooled down in a cold room for ease of handling and decapitated and double-pithed under dim red light. The retinas were removed from the hemisected eyeballs and placed into buffer A (67 mM sodium phosphate, pH 7.0, 2.7 mM KCl, 0.5 mM MgCl2, 1.0 mM CaCl2, 0.1 mM EDTA, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride) on ice immediately. After homogenization with an 18 gauge needle several times, the photoreceptor outer segments were purified by a two-step sucrose floatation first in buffer B (45% sucrose in buffer A) and then in buffer C (37% sucrose in buffer A) [15]. The ratio of OD280/OD521 for purified outer segments is between 3 and 3.5, which is near the value obtained for the purified pigments (2.8), indicating that the purified photoreceptor outer segments are quite pure. The prepared photoreceptor outer segments were stored in buffer A at −70°C until use.
2.2. Determination of lipid class composition Total lipids from gecko photoreceptor outer segments were extracted twice with chloroform/methanol according to Blight and Dyer [3] and washed 1× by the method of Folch et al. [9]. The lipid extracts were resolved into lipid classes by two-dimensional, threestep thin-layer chromatography (TLC) using a KG silica gel plate (10 ×20 cm, Sigma) as described by Choe and Anderson [6], with slight modifications to improve the resolution: hexane/diethyl ether (140:35, by vol.) for the second step separation and chloroform/ methanol/acetone/acetic acid/H2O (80/26/30/24/8, by vol.) for the third step. After visualizing the silica gel plates using 2, 7-dichlorofluorescein and UV light, spots on the plates containing individual lipid classes were scraped into glass tubes. Transmethylation of fatty acids was accomplished with 1 ml of 14% anhydrous BF3/methanol at 100°C for 20 mins in the presence of known amounts of internal standards (C15:0, C17:0 and C21:0). Fatty acid methyl esters (FAMEs) were extracted 3× with hexane and
the combined hexane extracts were washed 1× with water. FAMEs were dried under nitrogen, resuspended in 10–30 ml of nonane, and analyzed on a Varian 3500 gas–liquid chromatograph (Walnut Creek, CA) equipped with a DB-225 capillary column (30 m×0.25 mm I.D.; J & W Scientific, Folsom, CA) using the procedures of Chen and Anderson [5]. Chromatographic data were captured and integrated as area units using a PE Nelson Interface and further processed with the Turbochrom program (PE Nelson, Cupertino, CA). The mole quantity of each fatty acid was calculated using both C17:0 and C21:0 as internal standards. The mole quantity of the individual lipid class was calculated from total fatty acids recovered based upon fatty acid/lipid molar ratio of 1 for free fatty acids (FFA), sphingomyelin (SPH), and lyso-phospholipids; 2 for diacyl glyceride and diacyl phospholipids; and 3 for triglycerides (TG) and normalized for all classes analyzed to obtain lipid class composition. We also analyzed the lipid and fatty acid composition of bovine and frog photoreceptor outer segment membranes with the same method described above. This was done so we could minimize variations and facilitate comparison of the data.
3. Results The lipid class composition of gecko photoreceptor outer segment membranes is shown in Table 1. Three major phospholipids, PC, PE and PS, make up more than 80% of the lipids analyzed. The next most abundant lipid class found is free fatty acids (7.8%), whose value is between those determined for human (2.1%) Table 1 Lipid class composition (mol.%) of gecko photoreceptor outer segment membranes (values are means 9 standard deviation of four independent preparations and analyses) Lipid
Mol.%
PC PE PS PI DG PA TG FFA SPH L-PC L-PE L-PS
39.9 91.2 25.3 90.8 17.7 9 0.8 1.1 9 0.8 2.1 90.1 0.5 90.1 0.7 90.4 7.8 9 1.4 0.7 9 0.1 1.3 90.3 2.2 90.9 0.7 90.2
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; DG, diacyl glyceride; PA, phosphatidic acid; TG, triglyceride; FFA, free fatty acid; SPH, sphingomyelin; L-, lyso-phospholipid.
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Table 2 Fatty acid composition (mol.%) of PC, PE, PS, DG and PI from gecko photoreceptor outer segment membranes (mean 9S.D., n =4) Fatty Acid
PC
PE
PS
DG
PI
14:0 16:0 18:0 20:0 22:0 24:0 Subtotal
1.1 90.1 22.991.8 3.5 90.3 0.1 90.0 T T 27.6 92.3
T* 2.8 90.2 34.6 90.4 0.5 90.0 0.1 90.0 T 38.1 90.6
T 0.3 9 0.1 12.3 93.2 0.3 90.0 0.3 90.1 0.1 9 0.0 13.3 93.4
T 9.5 90.3 25.9 9 0.7 0.5 9 0.5 0.1 9 0.1 0.1 90.1 36.0 9 1.0
T 4.9 9 2.0 34.3 95.2 0.9 90.3 0.4 90.3 T 40.6 93.8
16:1 18:1 20:1 22:1 24:1 Subtotal
3.790.5 9.4 91.2 0.2 90.0 T T 13.3 91.7
0.6 90.1 6.590.4 0.3 90.1 0.1 90.0 T 7.5 90.5
0.4 9 0.2 3.9 91.7 0.3 9 0.1 0.3 90.1 0.1 9 0.0 4.8 9 1.9
1.4 90.1 9.0 9 1.9 0.2 9 0.2 0.5 9 0.5 0.4 90.3 11.4 92.9
2.7 9 0.6 24.9 96.9 0.6 90.4 0.9 90.9 T 29.1 98.5
18:2n-6 18:3n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 24:4n-6 24:5n-6 Subtotal
0.990.1 T 0.2 90.0 2.0 90.2 0.1 90.0 0.4 90.3 T T 3.7 90.5
0.8 90.1 T 0.3 90.0 6.990.4 0.6 90.1 0.7 90.4 0.2 90.0 0.1 90.1 9.5 90.8
0.4 9 0.1 T 0.3 9 0.0 1.8 90.3 1.2 90.1 1.4 9 0.8 1.1 90.5 0.5 9 0.3 6.6 9 1.2
1.8 90.4 T 2.0 9 0.4 23.6 9 1.0 0.4 9 0.0 0.8 9 0.2 T T 28.5 90.7
3.1 9 0.6 T 5.1 93.1 12.3 97.6 0.7 90.7 0.5 90.5 T T 21.6 96.5
18:3n-3 20:5n-3 22:5n-3 22:6n-3 24:5n-3 24:6n-3 Subtotal
T 0.3 90.1 T 54.99 3.7 0.19 0.0 0.19 0.0 55.49 3.6
T 0.6 90.2 0.2 90.2 42.59 0.9 0.39 0.1 1.39 0.0 44.99 0.7
T 0.2 9 0.1 T 62.7 9 3.4 2.2 9 0.1 10.2 9 0.7 75.3 9 4.1
T 2.89 0.6 0.6 9 0.2 20.69 4.0 T T 24.1 9 3.7
T 1.8 91.0 0.5 90.2 6.2 92.7 T 0.1 90.2 8.6 92.6
* T, trace element (B0.1%).
and goldfish (21.0%, see Ref. [8]). Minor lipids such as PI and DG comprise B 3% of the total lipids. Table 2 shows the fatty acid composition of PC, PE, PS, PI and DG in gecko photoreceptor outer segment membranes. The fatty acid compositions of the three major phospholipids, PC, PE and PS, are characterized by rather high levels of docosahexaenoic acid (22:6n-3). More than half (55%) of the PC molecules contain 22:6n-3, while saturated and monoenoic species are found in 40% of the total fatty acids. PE is also enriched in 22:6n-3 (43%) as well as 18:0 (35%). PS has the highest percentage of 22:6n-3 (63%) and 24:6n-3 (10%) with a total of 75% n-3 fatty acids among all the lipids analyzed. In bovine and rat photoreceptor outer segment membranes, the fatty acid compositions of the minor lipids such as DG and PI are quite different from those found in PC, PE or PS in richness of 20:4n-6 and 22:6n-3 [19]. The 20:4n-6 level is higher than 22:6n-3 contrary to what is in PE, PC and PS. Our results (Table 2) indicate that the fatty acid composition of DG and PI in gecko photoreceptor membranes is more similar to each other than to PE, PC and PS. Both contain relatively higher
levels of 20:4n-6 (12–24%) and lower levels of 22:6n-3 (6–21%). When compared with the lipid and fatty acid compositions of photoreceptor outer segment membranes from species such as bovine and frog, gecko shows several interesting differences.
3.0.1. More negati6ely-charged phospholipids Although the PC level is slightly higher than in bovine and is comparable with that in frog outer segment membranes (Table 3), a significantly lower level of Table 3 Comparison of diacyl lipid class composition (mol.%) of gecko, bovine and frog photoreceptor outer segment membranes (n =4) Lipid
Gecko
Bovine
Frog
PC PE PS PI DG PA
46.1 29.2 20.4 1.3 2.4 0.6
39.8 40.7 12.5 4.0 2.5 0.6
46.2 39.1 9.1 0.8 4.3 0.4
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Table 4 Relative content (mol.%) of docosahexaenoic acid (DHA, 22:6n-3) in three major phospholipids from gecko, bovine and frog outer segment membranes (n =4 for gecko, bovine and frog) Lipid
Gecko
Bovine
Frog
PC PE PS
54.9 42.5 62.8
25.3 43.6 42.4
31.2 57.8 62.1
PE and a much higher level of PS were found. Since PS is negatively charged, the net charge of the phospholipids of gecko photoreceptors will be more negative than in bovine or frog.
3.0.2. Higher DHA content Table 4 shows the comparison of DHA level among gecko, bovine and frog photoreceptor outer segment membranes. The DHA content is clearly higher in gecko for PC (55 vs 25% in bovine or 31% in frog). The level of DHA in PS of gecko photoreceptor outer segment membranes is the highest ever found; its level (63%) is comparable with what is found in frog (62%) but clearly higher than bovine (42%). Interestingly, these two phospholipids with higher DHA content also make up a larger fraction of the total lipid composition as shown in Table 3 (67 in gecko vs 52% in bovine or 55% in frog). On the other hand, gecko photoreceptor outer membrane has less PE while the DHA content of PE remains roughly the same as in bovine (43 – 44%). On average, phospholipids in gecko photoreceptor outer segment membranes contain 51% of DHA as compared with 33% in bovine and 43% in frog.
kinetics of Meta I/Meta II intermediates of bovine rhodopsin suggest that the apparent pKa of the equilibrium increases in the PS/PC reconstituted membranes. This has been attributed to the increased concentration of protons next to the membrane, which is modulated by the surface potential. Interestingly, PE has a similar effect on the equilibrium of Meta I/Meta II when bovine rhodopsin was reconstituted with PC/ PE. Flash photolysis studies by Liang et al. [16] on the rate of formation and decay of metarhodopsin of gecko P521 solubilized in digitonin revealed that gecko P521 did not show a significantly distinct kinetic behavior compared with bovine rhodopsin; however, the apparent pKa of the meta II formation was 8.7, higher than the value (6.4) for bovine rhodopsin [20]. A more negatively charged membrane environment may modulate the Meta I/Meta II equilibrium toward Meta II. Another interesting finding about gecko photoreceptor outer membrane lipids is their unusually high DHA content. A high percentage of lipids with DHA in photoreceptor has been suggested to be important for the functioning of photoreceptors from diet-deficiency studies in rats [10]. It has been suggested that the leakage of Na + through rod outer segment membrane is related to DHA-containing phospholipids and cholesterol [13]. Metarhodopsin II formation is also found to be favored in the presence of lipids containing unsaturated fatty acid such as DHA [18]. In summary, the high DHA content in phospholipids from gecko photoreceptor outer segment membranes may reflect a specific requirement of the lipid environment for maintaining the proper structure and function of cone-type pigments.
Acknowledgements 4. Discussion Our analysis of the phospholipid composition of gecko photoreceptor outer segment membranes shows that it is very different from the other vertebrates examined so far. Over 90% of the visual pigment is the cone-type visual pigment P521 and thus the composition of the photoreceptor outer segment membranes is dominated by lipids in membranes containing this pigment. Compared with bovine membranes, gecko photoreceptor outer segment membranes contain :16% more PC, 28% less PE and 63% more PS. The increase in negatively charged PS should render the surface potential more negative and possibly change some properties of the visual pigment. If we assume the same ratio of phospholipid-to-visual pigment as found in bovine photoreceptors (65:1) [14,17], then each gecko P521 pigment molecule would be associated with three more negative charges from phospholipids than bovine rhodopsin. Previous studies by Gibson and Brown [12] on the
We thank Dr Li Tang for helping the preparation of the gecko photoreceptor outer segments. This work is supported in part by NIH (EY01323 to T.G.E.and EY00871 and EY04149 to R.E.A.); The Foundation Fighting Blindness (Baltimore, MD); Research to Prevent Blindness. (New York, NY); Presbyterian Health Foundation (Oklahoma City, OK); and Samuel Roberts Noble Foundation (Ardmore, OK). R.E.A. is a Senior Scientific Investigator of Research to Prevent Blindness.
References [1] Anderson RE. Lipid of ocular tissues IV: A comparison of the phospholipids from the retina of six mammalian species. Exp Eye Res 1970;10:339 – 44. [2] Bazan NG, Escalante MS, Careaga MM, Bazan HEP, Giusto NM. High content of 22: 6 (docosahexaenoate) and active [2-3H] glycerol metabolism of phosphatidic acid from photoreceptor membrane. Biochim Biophys Acta 1982;712:702 – 16.
C. Yuan et al. / Comparati6e Biochemistry and Physiology, Part B 120 (1998) 785–789 [3] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–7. [4] Boesze-Battaglia K, Albert AD. Cholesterol modulation of photoreceptor function in bovine rod outer segments. J Biol Chem 1990;265:20727– 30. [5] Chen H, Anderson RE. Lipids of frog retinal pigment epithelium: comparison with rod outer segments, retina, plasma and red blood cells. Curr Eye Res 1992;11:793–800. [6] Choe HG, Anderson RE. Unique molecular species composition of glycerolipid of frog rod outer segments. Exp Eye Res 1990;51: 159 – 65. [7] Crescitelli F. In: Crescitelli F, editor. Handbook of Sensory Physiology. Berlin: Springer, 1977:391–449. [8] Fliesler SJ, Anderson RE. Chemistry and metabolism of lipids in the vertebrate retina. Prog Lipid Res 1983;22:79–131. [9] Folch J, Lees M, Stanley GH. A simple method for the isolation and purification of total lipids form animal tissues. J Biol Chem 1957;226:497 – 509. [10] Futterman S, Downer JC, Hendrickson A. Effect of essential fatty acid deficiency on the fatty acid composition, morphology and electroretinographic response of the retina. Investig Ophthalmol 1971;10:151 –6. [11] Gibson NJ, Brown MF. Role of phosphatidylserine in the MI – MII equilibrium of rhodopsin. Biochim Biophys Res Commun 1991;176:915 – 21. [12] Gibson NJ, Brown MF. Lipid headgroup and acyl chain composition modulate the MI–MII equilibrium of rhodopsin in recombinant membranes. Biochemistry 1993;32:2438–54.
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[13] Hendricks TH, Klompmakers AA, Daemen FJM, Bonting SL. Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayer composed of retinal and rod outer segment lipids. Biochim Biophys Acta 1976;433:271 – 81. [14] Hubbell WL. Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes. Biophys J 1990;57:99 – 108. [15] Jonas RE, Yuan C, Ebrey TG. Improved large-scale purification of transducin, and its a and bg subunits from frozen retinas. Prep Biochem 1994;24:279 – 88. [16] Liang J, Govindjee R, Ebrey TG. Metarhodopsin intermediates of the gecko cone pigment P521. Biochemistry 1993;32:14187– 93. [17] Miljanich GP, Nemes DL, Dratz EA. The asymmetric transmembrane distribution of phosphatidylethanolamine, phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane. J Membr Biol 1981;60:249 – 55. [18] Mitchell D, Straume M, Miller JL, Litman BJ. Role of sn-1-saturated, sn-2 polyunsaturated phospholipids in control of membrane receptor conformational equilibrium, effects of cholesterol and acyl chain unsaturation on the metarhodopsin Imetarhodopsin II equilibrium. Biochemistry 1992;31:662–70. [19] Stinson AM, Wiegand RD, Anderson RE. Fatty acid and molecular species compositions of phospholipids and diacylglycerols from rat retinal membrane. Exp Eye Res 1991;52:213 –8. [20] Weitz CJ, Nathans J. Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II. Neuron 1992;8:465 – 72.
The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes Ching Yuan a, Huiming Chen c,e,f, Robert E. Anderson c,d,e,f, Osamu Kuwata b, Thomas G. Ebrey a,b,* b
a Center for Biophysics and Computational Biology, Uni6ersity of Illinois at Urbana-Champaign, Urbana, IL, USA Department of Cell and Structural Biology, Uni6ersity of Illinois at Urbana-Champaign, 601 South Goodwin A6enue, Urbana, IL 61801, USA c Department of Ophthalmology, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA d Biochemistry & Molecular Biology, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA e Oklahoma Center for Neuroscience, Uni6ersity of Oklahoma Health Sciences Center, Oklahoma City, OK, USA f Dean A. McGee Eye Institute, Oklahoma City, OK, USA
Received 5 March 1998; received in revised form 22 June 1998; accepted 1 July 1998
Abstract This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Cell membrane; Lipid composition; Gecko; Retina; Photoreceptor; Cone-type visual pigment; Docosahexaenoic acid; Phospholipid
1. Introduction The composition and molecular species of the lipids in photoreceptor outer segment membranes have been investigated extensively for several types of vertebrates, mostly mammals and amphibians [1,2,8,19]. A remarkable similarity was observed for the various phospholipid species: PC, 40 – 50%; PE, 30 – 35%; PS, 5 –15%; phosphatidylinositol (PI), 3 – 6%; diacyl glyceride (DG), 1 – 2%; and phosphatidic acid (PA), less than 1%. The phospholipid composition is known to influence the kinetics and equilibrium of the Meta I/Meta II intermediates of rhodopsin [11,12] and the kinetics and extent of phosphodiesterase activation [4]. * Corresponding author. Tel.: +1 217 3332015; fax: + 1 217 2446615; e-mail: [email protected] 0305-0491/98/$19.00 © 1998 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10079-2
The retinas of the animal species studied so far have only a small percentage of their total visual pigments as cone pigments. It would be interesting to know whether the lipid composition of photoreceptor cells with conetype pigments is different from those with rod-type pigments and whether cone-type pigments require a distinct lipid environment for their proper function compared to rhodopsin. The gecko retina contains almost exclusively rod shaped photoreceptor cells. However, more than 90% of the visual pigment in these photoreceptor cells is a cone-type pigment, P521 [7]. In this report we determined the fatty acid and phospholipid class composition of gecko photoreceptor outer segment membranes. Our results show that its composition is somewhat different from what is found in photoreceptors containing rod-type pigments. In gecko
786
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photoreceptors, there is more negatively charged phospholipid, PS, compared to bovine and frog. Furthermore, a surprisingly high percentage of DHA is found in PC and PS isolated from gecko photoreceptor membranes. The molar percentage of these two phospholipid species are also higher in the gecko photoreceptor outer segment membranes.
2. Materials and methods
2.1. Preparation of gecko photoreceptor outer segments Tokay geckos from WestCoast Reptile (Fullerton, CA) were dark-adapted for at least 12 h at room temperature. The animals were then cooled down in a cold room for ease of handling and decapitated and double-pithed under dim red light. The retinas were removed from the hemisected eyeballs and placed into buffer A (67 mM sodium phosphate, pH 7.0, 2.7 mM KCl, 0.5 mM MgCl2, 1.0 mM CaCl2, 0.1 mM EDTA, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride) on ice immediately. After homogenization with an 18 gauge needle several times, the photoreceptor outer segments were purified by a two-step sucrose floatation first in buffer B (45% sucrose in buffer A) and then in buffer C (37% sucrose in buffer A) [15]. The ratio of OD280/OD521 for purified outer segments is between 3 and 3.5, which is near the value obtained for the purified pigments (2.8), indicating that the purified photoreceptor outer segments are quite pure. The prepared photoreceptor outer segments were stored in buffer A at −70°C until use.
2.2. Determination of lipid class composition Total lipids from gecko photoreceptor outer segments were extracted twice with chloroform/methanol according to Blight and Dyer [3] and washed 1× by the method of Folch et al. [9]. The lipid extracts were resolved into lipid classes by two-dimensional, threestep thin-layer chromatography (TLC) using a KG silica gel plate (10 ×20 cm, Sigma) as described by Choe and Anderson [6], with slight modifications to improve the resolution: hexane/diethyl ether (140:35, by vol.) for the second step separation and chloroform/ methanol/acetone/acetic acid/H2O (80/26/30/24/8, by vol.) for the third step. After visualizing the silica gel plates using 2, 7-dichlorofluorescein and UV light, spots on the plates containing individual lipid classes were scraped into glass tubes. Transmethylation of fatty acids was accomplished with 1 ml of 14% anhydrous BF3/methanol at 100°C for 20 mins in the presence of known amounts of internal standards (C15:0, C17:0 and C21:0). Fatty acid methyl esters (FAMEs) were extracted 3× with hexane and
the combined hexane extracts were washed 1× with water. FAMEs were dried under nitrogen, resuspended in 10–30 ml of nonane, and analyzed on a Varian 3500 gas–liquid chromatograph (Walnut Creek, CA) equipped with a DB-225 capillary column (30 m×0.25 mm I.D.; J & W Scientific, Folsom, CA) using the procedures of Chen and Anderson [5]. Chromatographic data were captured and integrated as area units using a PE Nelson Interface and further processed with the Turbochrom program (PE Nelson, Cupertino, CA). The mole quantity of each fatty acid was calculated using both C17:0 and C21:0 as internal standards. The mole quantity of the individual lipid class was calculated from total fatty acids recovered based upon fatty acid/lipid molar ratio of 1 for free fatty acids (FFA), sphingomyelin (SPH), and lyso-phospholipids; 2 for diacyl glyceride and diacyl phospholipids; and 3 for triglycerides (TG) and normalized for all classes analyzed to obtain lipid class composition. We also analyzed the lipid and fatty acid composition of bovine and frog photoreceptor outer segment membranes with the same method described above. This was done so we could minimize variations and facilitate comparison of the data.
3. Results The lipid class composition of gecko photoreceptor outer segment membranes is shown in Table 1. Three major phospholipids, PC, PE and PS, make up more than 80% of the lipids analyzed. The next most abundant lipid class found is free fatty acids (7.8%), whose value is between those determined for human (2.1%) Table 1 Lipid class composition (mol.%) of gecko photoreceptor outer segment membranes (values are means 9 standard deviation of four independent preparations and analyses) Lipid
Mol.%
PC PE PS PI DG PA TG FFA SPH L-PC L-PE L-PS
39.9 91.2 25.3 90.8 17.7 9 0.8 1.1 9 0.8 2.1 90.1 0.5 90.1 0.7 90.4 7.8 9 1.4 0.7 9 0.1 1.3 90.3 2.2 90.9 0.7 90.2
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; DG, diacyl glyceride; PA, phosphatidic acid; TG, triglyceride; FFA, free fatty acid; SPH, sphingomyelin; L-, lyso-phospholipid.
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Table 2 Fatty acid composition (mol.%) of PC, PE, PS, DG and PI from gecko photoreceptor outer segment membranes (mean 9S.D., n =4) Fatty Acid
PC
PE
PS
DG
PI
14:0 16:0 18:0 20:0 22:0 24:0 Subtotal
1.1 90.1 22.991.8 3.5 90.3 0.1 90.0 T T 27.6 92.3
T* 2.8 90.2 34.6 90.4 0.5 90.0 0.1 90.0 T 38.1 90.6
T 0.3 9 0.1 12.3 93.2 0.3 90.0 0.3 90.1 0.1 9 0.0 13.3 93.4
T 9.5 90.3 25.9 9 0.7 0.5 9 0.5 0.1 9 0.1 0.1 90.1 36.0 9 1.0
T 4.9 9 2.0 34.3 95.2 0.9 90.3 0.4 90.3 T 40.6 93.8
16:1 18:1 20:1 22:1 24:1 Subtotal
3.790.5 9.4 91.2 0.2 90.0 T T 13.3 91.7
0.6 90.1 6.590.4 0.3 90.1 0.1 90.0 T 7.5 90.5
0.4 9 0.2 3.9 91.7 0.3 9 0.1 0.3 90.1 0.1 9 0.0 4.8 9 1.9
1.4 90.1 9.0 9 1.9 0.2 9 0.2 0.5 9 0.5 0.4 90.3 11.4 92.9
2.7 9 0.6 24.9 96.9 0.6 90.4 0.9 90.9 T 29.1 98.5
18:2n-6 18:3n-6 20:3n-6 20:4n-6 22:4n-6 22:5n-6 24:4n-6 24:5n-6 Subtotal
0.990.1 T 0.2 90.0 2.0 90.2 0.1 90.0 0.4 90.3 T T 3.7 90.5
0.8 90.1 T 0.3 90.0 6.990.4 0.6 90.1 0.7 90.4 0.2 90.0 0.1 90.1 9.5 90.8
0.4 9 0.1 T 0.3 9 0.0 1.8 90.3 1.2 90.1 1.4 9 0.8 1.1 90.5 0.5 9 0.3 6.6 9 1.2
1.8 90.4 T 2.0 9 0.4 23.6 9 1.0 0.4 9 0.0 0.8 9 0.2 T T 28.5 90.7
3.1 9 0.6 T 5.1 93.1 12.3 97.6 0.7 90.7 0.5 90.5 T T 21.6 96.5
18:3n-3 20:5n-3 22:5n-3 22:6n-3 24:5n-3 24:6n-3 Subtotal
T 0.3 90.1 T 54.99 3.7 0.19 0.0 0.19 0.0 55.49 3.6
T 0.6 90.2 0.2 90.2 42.59 0.9 0.39 0.1 1.39 0.0 44.99 0.7
T 0.2 9 0.1 T 62.7 9 3.4 2.2 9 0.1 10.2 9 0.7 75.3 9 4.1
T 2.89 0.6 0.6 9 0.2 20.69 4.0 T T 24.1 9 3.7
T 1.8 91.0 0.5 90.2 6.2 92.7 T 0.1 90.2 8.6 92.6
* T, trace element (B0.1%).
and goldfish (21.0%, see Ref. [8]). Minor lipids such as PI and DG comprise B 3% of the total lipids. Table 2 shows the fatty acid composition of PC, PE, PS, PI and DG in gecko photoreceptor outer segment membranes. The fatty acid compositions of the three major phospholipids, PC, PE and PS, are characterized by rather high levels of docosahexaenoic acid (22:6n-3). More than half (55%) of the PC molecules contain 22:6n-3, while saturated and monoenoic species are found in 40% of the total fatty acids. PE is also enriched in 22:6n-3 (43%) as well as 18:0 (35%). PS has the highest percentage of 22:6n-3 (63%) and 24:6n-3 (10%) with a total of 75% n-3 fatty acids among all the lipids analyzed. In bovine and rat photoreceptor outer segment membranes, the fatty acid compositions of the minor lipids such as DG and PI are quite different from those found in PC, PE or PS in richness of 20:4n-6 and 22:6n-3 [19]. The 20:4n-6 level is higher than 22:6n-3 contrary to what is in PE, PC and PS. Our results (Table 2) indicate that the fatty acid composition of DG and PI in gecko photoreceptor membranes is more similar to each other than to PE, PC and PS. Both contain relatively higher
levels of 20:4n-6 (12–24%) and lower levels of 22:6n-3 (6–21%). When compared with the lipid and fatty acid compositions of photoreceptor outer segment membranes from species such as bovine and frog, gecko shows several interesting differences.
3.0.1. More negati6ely-charged phospholipids Although the PC level is slightly higher than in bovine and is comparable with that in frog outer segment membranes (Table 3), a significantly lower level of Table 3 Comparison of diacyl lipid class composition (mol.%) of gecko, bovine and frog photoreceptor outer segment membranes (n =4) Lipid
Gecko
Bovine
Frog
PC PE PS PI DG PA
46.1 29.2 20.4 1.3 2.4 0.6
39.8 40.7 12.5 4.0 2.5 0.6
46.2 39.1 9.1 0.8 4.3 0.4
C. Yuan et al. / Comparati6e Biochemistry and Physiology, Part B 120 (1998) 785–789
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Table 4 Relative content (mol.%) of docosahexaenoic acid (DHA, 22:6n-3) in three major phospholipids from gecko, bovine and frog outer segment membranes (n =4 for gecko, bovine and frog) Lipid
Gecko
Bovine
Frog
PC PE PS
54.9 42.5 62.8
25.3 43.6 42.4
31.2 57.8 62.1
PE and a much higher level of PS were found. Since PS is negatively charged, the net charge of the phospholipids of gecko photoreceptors will be more negative than in bovine or frog.
3.0.2. Higher DHA content Table 4 shows the comparison of DHA level among gecko, bovine and frog photoreceptor outer segment membranes. The DHA content is clearly higher in gecko for PC (55 vs 25% in bovine or 31% in frog). The level of DHA in PS of gecko photoreceptor outer segment membranes is the highest ever found; its level (63%) is comparable with what is found in frog (62%) but clearly higher than bovine (42%). Interestingly, these two phospholipids with higher DHA content also make up a larger fraction of the total lipid composition as shown in Table 3 (67 in gecko vs 52% in bovine or 55% in frog). On the other hand, gecko photoreceptor outer membrane has less PE while the DHA content of PE remains roughly the same as in bovine (43 – 44%). On average, phospholipids in gecko photoreceptor outer segment membranes contain 51% of DHA as compared with 33% in bovine and 43% in frog.
kinetics of Meta I/Meta II intermediates of bovine rhodopsin suggest that the apparent pKa of the equilibrium increases in the PS/PC reconstituted membranes. This has been attributed to the increased concentration of protons next to the membrane, which is modulated by the surface potential. Interestingly, PE has a similar effect on the equilibrium of Meta I/Meta II when bovine rhodopsin was reconstituted with PC/ PE. Flash photolysis studies by Liang et al. [16] on the rate of formation and decay of metarhodopsin of gecko P521 solubilized in digitonin revealed that gecko P521 did not show a significantly distinct kinetic behavior compared with bovine rhodopsin; however, the apparent pKa of the meta II formation was 8.7, higher than the value (6.4) for bovine rhodopsin [20]. A more negatively charged membrane environment may modulate the Meta I/Meta II equilibrium toward Meta II. Another interesting finding about gecko photoreceptor outer membrane lipids is their unusually high DHA content. A high percentage of lipids with DHA in photoreceptor has been suggested to be important for the functioning of photoreceptors from diet-deficiency studies in rats [10]. It has been suggested that the leakage of Na + through rod outer segment membrane is related to DHA-containing phospholipids and cholesterol [13]. Metarhodopsin II formation is also found to be favored in the presence of lipids containing unsaturated fatty acid such as DHA [18]. In summary, the high DHA content in phospholipids from gecko photoreceptor outer segment membranes may reflect a specific requirement of the lipid environment for maintaining the proper structure and function of cone-type pigments.
Acknowledgements 4. Discussion Our analysis of the phospholipid composition of gecko photoreceptor outer segment membranes shows that it is very different from the other vertebrates examined so far. Over 90% of the visual pigment is the cone-type visual pigment P521 and thus the composition of the photoreceptor outer segment membranes is dominated by lipids in membranes containing this pigment. Compared with bovine membranes, gecko photoreceptor outer segment membranes contain :16% more PC, 28% less PE and 63% more PS. The increase in negatively charged PS should render the surface potential more negative and possibly change some properties of the visual pigment. If we assume the same ratio of phospholipid-to-visual pigment as found in bovine photoreceptors (65:1) [14,17], then each gecko P521 pigment molecule would be associated with three more negative charges from phospholipids than bovine rhodopsin. Previous studies by Gibson and Brown [12] on the
We thank Dr Li Tang for helping the preparation of the gecko photoreceptor outer segments. This work is supported in part by NIH (EY01323 to T.G.E.and EY00871 and EY04149 to R.E.A.); The Foundation Fighting Blindness (Baltimore, MD); Research to Prevent Blindness. (New York, NY); Presbyterian Health Foundation (Oklahoma City, OK); and Samuel Roberts Noble Foundation (Ardmore, OK). R.E.A. is a Senior Scientific Investigator of Research to Prevent Blindness.
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