Molecular Vision 2000; 6:109-115 <http://www.molvis.org/molvis/v6/a15/> Received 23 March 2000 | Accepted 24 May 2000 | Published 27 June 2000

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Mass spectrometric analysis of rhodopsin from light damaged rats

Zs. Ablonczy,^1,2 D. R. Knapp,¹ R. Darrow,³ D. T. Organisciak,³ Rosalie K. Crouch¹
 
 

¹Medical University of South Carolina, Charleston, SC; ²Eötvös University, Budapest, Hungary; ³Petticrew Research Laboratory, Wright State University, Dayton, OH

Correspondence to: Rosalie K. Crouch, Ph.D., Department of Ophthalmology, Medical University of South Carolina, 707 SEI 167 Ashley Avenue, Charleston, SC, 29425; Phone: (843) 792-3031; FAX: (843) 792-5110; email: crouchrk@musc.edu

Abstract

Purpose: It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level.

Methods: Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation.

Results: The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported.

Conclusions: Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.

Introduction

In 1966, Noell et al. first described visible light induced retinal degeneration in rats [1]. His study and subsequent work indicate that the action spectrum of this "light damage" is that of the absorption spectrum of rhodopsin [1,2]. It is now recognized that light damage is species-specific and is dependent on the duration and intensity of the damaging light, the nature of the absorbing chromophores in the retina [1,3-7], and the age and environmental history of the animals [8-11] as well as their genetic characteristics [12,13]. There is compelling evidence that extensive bleaching of rhodopsin induces oxidative stress in the retina that can affect the entire cell. Long term exposure may lead to lipid peroxidation of rod outer segments [14], but morphologic changes manifest in the inner segment as well [15]. Oxidative and apoptotic light induced fragmentation of DNA has been reported [16-19], processes that are reduced by pretreatment of the animals with antioxidants [20-24]. It is not clear, however, if the bleaching of rhodopsin that initiates this damage has any oxidative, truncative, or other effects on the rhodopsin protein itself. The purpose of this study was to examine, by mass spectrometric methods, the structure of rhodopsin from control and light damaged animals.

Over the past several years, mass spectrometry has been increasingly applied to studies of protein structure [25-27]. Most commonly, mass spectrometric methods have been used with soluble proteins that generally yield soluble and chromatographically well-behaved peptide cleavage products. Integral membrane proteins have posed serious challenges for analysis by mass spectrometric methods, but methodology has now been developed that allows observation of the entire sequence of rhodopsin [28]. In the work reported here, we applied this method to the study of rhodopsin derived from rod outer segments of light damaged rats.

Light-stimulated phosphorylation of rhodopsin was first described 28 years ago [29]. Upon irradiation, rhodopsin is converted to an activated state. The deactivation process involves phosphorylation of the protein with rhodopsin kinase [30], and phosphorylation promotes arrestin binding, which prevents the further activation of the visual cascade. In vitro, it has been demonstrated that bleached rhodopsin can incorporate up to nine moles of phosphate per mole of protein [31]. The most probable phosphorylation sites are the hydroxylamino acids that lie in the C terminal region of the protein [32-34]. Previous in vitro mass spectrometric experiments identified single sites of rhodopsin phosphorylation [35,36] while in vivo studies have shown multiple phosphorylation sites on the protein [37,38]. In this study, we identified three major sites of phosphorylation on the rhodopsin of light damaged rats.

Methods

Animal care and rhodopsin extraction

Normal albino Sprague-Dawley (SD) rats, Royal College of Surgeons (RCS) dystrophic rats (Harlan Inc., Indianapolis, IN) and heterozygous rats having a P23H mutation in rhodopsin (line 3, from Dr. M. LaVail, UCSF School of Medicine, San Francisco, CA) were reared in darkness or in a weak cyclic light environment consisting of 12 h of 20-40 lux light per day. The RCS dystrophic animals were 24 days of age, the P23H mutated heterozygotes were 38-50 days, and the normal SD rats 60-75 days when sacrificed. Animals were exposed to intense visible light for various periods of time with unexposed controls being kept under normal conditions. The animals reared in cyclic light were dark adapted for 16 h before sacrifice or light exposure. The length of exposure for P23H mutated rats was 4 h for the cyclic and 2 h for the dark reared animal. The exposure time for normal SD rats was 8-24 h. Only non-light exposed RCS rats ("control RCS rats") were used in these experiments. The chambers used for light exposure and the exposure conditions have previously been described [39]. Following light exposure some animals were kept in darkness for 24 h while others were sacrificed immediately. All rats were sacrificed in red light in carbon dioxide saturated chambers and their retinas excised within 2 min of death. Rod outer segments were immediately isolated by sucrose gradient ultracentrifugation [24] and rhodopsin prepared by the method of McDowell and Kühn [40]. The final purified sample, from 8-12 pooled retinae, contained approximately 175 mg of protein. Care was taken to purge all buffers with nitrogen and to store the extracted protein under argon to avoid oxidation. The samples were then wrapped in foil, and kept in the dark at -80 °C, then shipped on dry ice from the Wright State University to the Medical University of South Carolina for further analysis. The use of animals in this investigation conformed to the ARVO statement for the use of animals in research.

Generation of peptides for sequence analysis

The preparation of rhodopsin samples for mapping purposes followed the methods developed earlier for the mass spectrometric analysis of integral membrane proteins [28]. The purified rhodopsin samples were reduced with tributylphosphine, (Aldrich Chem. Co., Milwaukee, WI), alkylated with 4-vinylpyridine (Sigma Chem. Co., St. Louis, MO), and delipidated by precipitation in ethanol. The delipidated protein was cleaved with CNBr and then evaporated to dryness under vacuum. The dried fragment mixture was dissolved in 5 mL of solution equivalent to the initial HPLC gradient mobile phase (97.5% A and 2.5% B), where A is 0.05% aqueous TFA solvent and B is 2:1 isopropanol/acetonitrile containing 0.05% TFA. The sample was then loaded onto a 2.1 mm x 100 mm C4 Aquapore column (Perkin Elmer Brownlee Column, Bodman Industries, Aston, PA) at 400 mL/min of 2.5% B for 12.5 min. The peptides were eluted at a flow of 400 mL/min with a gradient of 2.5-60% B in 60 min and 60-97.5% B in 20 min. The HPLC was done by an HP1100 series HPLC instrument (Agilent Technologies, Palo Alto, CA). The C terminal fragment was dried under vacuum, dissolved in 50 mL of 9:1 100 mM ammonium bicarbonate at pH 8.3/acetonitrile and digested with 0.5 mg trypsin in 1 mM HCl overnight at 37 °C. The cleavage mixture was dried under vacuum and dissolved to yield 20 mL of solution equivalent to the initial HPLC gradient mobile phase (97.5% A and 2.5% B), where A is 0.1 M acetic acid and B is acetonitrile. The 20 mL sample was loaded onto a 0.32 mm x 150 mm C4 Intersil (Micro-Tech Scientific, Sunnyvale, CA) capillary column (at 5 mL/min of 2.5% B for 5 min) and the peptides eluted with a gradient of 2.5-97.5% B in 60 min.

For the determination of the amount and location of C terminal phosphorylation, samples were centrifuged for 15 min at 100,000 g, the supernatant was removed and the pellet suspended in 100 mL 10 mM Tris at pH 7.5. The protein was cleaved overnight at 37 °C with 50 ng endoproteinase Asp-N (Sigma Chemical Co., St. Louis, MO) at approximately 1/2000 enzyme to substrate ratio, dissolved in water. After centrifugation (15 min at 100,000 g), the supernatant was dried under vacuum and dissolved to yield 5 mL in the initial HPLC gradient mobile phase (97.5% A and 2.5% B), where A is 0.05% aqueous TFA solvent and B is acetonitrile containing 0.05% TFA. The 5 mL sample was loaded onto a 2.1 mm x 100 mm C4 Aquapore column (at 400 mL/min of 2.5% B for 12.5 min). The peptides were eluted at a flow of 200 mL/min with a gradient of 2.5-5% B in 5 min, 5-15% B in 10 or 20 min.

The glycosylated rhodopsin fragment was dried under vacuum, dissolved in 50 mM ammonium bicarbonate at pH 7.6 and treated for 30 min at 25 °C with N-glycosidase F and analyzed on MALDI-TOF (matrix assisted laser desorption ionization-time of flight mass spectrometry) instrument with the procedure below.

Mass spectrometry

The column effluent from the 2.1 mm HPLC columns was split and 10% of the flow was directed into the ESI source of a Finnigan LCQ ion trap mass spectrometer (Thermo Instrument Systems Inc., Waltham, MA). Capillary column effluents were not split. Data were acquired using LCQ version 1.2 software, details previously described [28]. MS (mass spectral) data were acquired by repetitive scanning with MS/MS data automatically acquired for the most intense precursor ion in each MS spectrum. The remaining 90% of the effluent was collected in 2 min fractions, dried under vacuum and stored for later use. These fractions were further analyzed with a Perseptive Voyager DE MALDI-TOF (delayed extraction MALDI-TOF) instrument (PE Biosystems, Foster City, CA) using a-cyano-4-hydroxycinnamic acid as matrix. The a-cyano-4-hydroxycinnamic acid was dissolved in 70% acetonitrile. The dried HPLC fractions were each dissolved in 4 mL of 70% acetonitrile. A 0.3 mL aliquot of the solution was spotted on the MALDI plate followed by 0.9 mL of the matrix solution. The spots were allowed to dry on the plate together with external standards for the calibration of the instrument.

Results

The rat rhodopsin samples were analyzed with two mass spectrometric techniques, ESI-MS (electronspray ionization-mass spectrometry) and MALDI-MS. In ESI-MS, the effluent of an HPLC column passes through a needle tip at high electrical potential, which results in a fine mist of highly charged droplets. After the evaporation of the solvents this yields multiply-charged peptide ions (with several different charge states), detected by the mass analyzer. The ion trap instrument is capable of storing a selected ion and fragmenting it by collision with inert gas. The fragmentation occurs mainly at peptide bonds. By detecting the masses of the different fragments, the amino acid sequence of 20-30 amino acid peptides can easily be reconstituted (MS/MS spectra). MALDI-MS requires mixing of the solution with an excess of small matrix molecules that absorb laser light. Upon excitation, the matrix molecules explode from the sample plate, ionizing the peptides in the solution. These peptides are then sorted by their time of flight in a vacuum tube by their mass/charge ratio and their abundance is detected. The MALDI technique resembles a protein gel but it is much more accurate.

Using these techniques, the rhodopsin from Sprague-Dawley rats was completely mapped with the exception of the two single amino acid fragments. Figure 1 shows the amino acid sequence and a two-dimensional model of rat rhodopsin, and Table 1 indicates its CNBr fragments.

MS/MS data were collected for 15 of the total 18 CNBr fragments. The two single amino acid fragments are not detectable with the instrument and no sequence data were collected for the glycosylated fragment 2. However fragment 2 was identifiable in the base peak chromatogram. The difference between the masses of fragment 2 from wild type rats and the P23H transgenics was also observed (+40 Da). In the case of P23H both mass peaks are present because of the two rhodopsins being present in the heterozygotes. MS/MS data were not obtained for fragment 2 due to the length of the peptide chain and the attached heterogeneous oligossacharides. By collecting 2 min fractions from the HPLC effluent, it was possible to find the exact location of fragment 2 in the gradient. These fractions were deglycosylated with N-glycosidase F and the peptide identified by the loss of 1096 Da, the mass for GlcNAc(Man)₃(GlcNAc)₂, the most abundant glycoform obtained by mass spectrometry earlier [41] (data not shown). Figure 2 shows LCQ base peak chromatograms for dark reared, light damaged animals and a cyclic reared control preparation, with the most abundant CNBr fragments indicated (fragments 3, 4, 5, 6, 7, 8, 14, 15, and 18). The fragments not indicated (fragments 2, 9, 10, 11, 12, 13, and 17) were found in the background. The base peak chromatogram shows the amount of the most abundant detected precursor ion eluting from the HPLC column at each time point. Since the detection limit was 400-2000 Da, only fragments 3 and 7 were detected as +1 charge state ions, the other fragments were detected in one of their higher charge states (see Table 1 for reference). The protein sample was not completely clean and there were contaminants also in the HPLC gradient (the most prominent one had a mass of 437 Da). As it can be seen in Figure 2, some of the fragments contained were oxidized tryptophans due to aged CNBr. The peaks are normalized to the most abundant ion in each chromatogram and therefore their relative amounts are not comparable.

The C-terminal fragment is also relatively large and has extensive posttranslational modifications (palmitylation and possible phosphorylation). The MS/MS data for this fragment were sufficient for the identification of the fragment but gave no information on the location of the modifications. Therefore, the collected fragments were digested with trypsin. The trypsin digest of fragment 18 provides three smaller peptide fragments which were easier to analyze. The MS/MS data for the first trypsin fragment of the C-terminal CNBr fragment 18 showed the amino acid at position 320 to be threonine (Figure 3). The amino acid is identified by the 101 Da mass difference between the b₂ and b₃ or y₅ and y₆ fragment ions. There is also a posttranslational modification on the same fragment. The loss of 238 Da from y₇ and some other ions indicated palmityl group loss, and the difference of 341 Da between y₃ and y₄ shows, that cysteine 322 was palmitylated. However, the mass ladder implies, that cysteine 323 was also palmitylated.

Apart from the above mentioned four CNBr fragments, the MS/MS data collected during a single HPLC run were sufficiently complete not only for the identification of the fragments but for the possible posttranslational modifications as well. The posttranslational modifications were found to be as previously reported for bovine rhodopsin. The first amino acid is acetylated, asparagines 2 and 15 are glycosylated, cysteines 322 and 323 are palmitylated, and, in the case of light adapted samples, there was phosphorylation on the C terminus. Figure 1 shows the sequence as well as the posttranslational modifications on the protein. No sequence modification between the light damaged and control animals was found. For the P23H samples, the appropriate mass shift was observed in fragment 2.

When light damaged animals were not dark-adapted before sacrifice, phosphorylation was observed. Phosphorylation was identified by a mass shift of +80 Da for HPO₃ (or its appropriate multiple for multiple phosphorylation) on the observed fragments. In order to identify the sites of phosphorylation, Asp-N digestion was used to cleave the last 19 amino acids of the protein [35,38]. Three main sites of phosphorylation were identified by MS/MS data. Threonine 336 as well as serines 334 and 338 were found to be monophosphorylated and their various combinations could be identified as the di- and triphosphorylated states. Tetraphosphorylation was also present, but the sites were not identified. Di- and triphosphorylation MS/MS data are difficult to analyze because of the low intensity of the mass peaks. There was an indication of possible phosphorylation on S 343 or T 342 on the doubly and triply phosphorylated species. T 340 was not found to be phosphorylated. Figure 4 shows comparison of the phosphorylation states of dim cyclic light reared light damaged/no dark-adaptation, light damaged/dark-adapted and control/dark-adapted animals. As can be seen, that light damaged and not dark-adapted samples contained multiple phosphorylation (identified by the +40 Da mass shift over the (M+2H)⁺² ion). The base peak chromatogram shows only triphosphorylation, but the mass spectrum reveals the very small amount of tetraphosphorylation. As seen by the other samples in the middle and on the bottom, dark-adaptation completely reversed the phosphorylation of the protein, even in the light damaged animals. It can also be seen that there is a very small peak (980 Da) in the control/dark adapted spectrum that indicates a small amount of phosphorylation.

Transgenic rats having a histidine substituted at the proline 23 position are known to be extremely light sensitive [42,43] and this mutation has been found in patients with one form of autosomal dominant retinitis pigmentosa [44]. We therefore examined the rhodopsin isolated from control and light exposed P23H transgenic rats. We did find the mutation at position 23 in the rod outer segment rhodopsin. However, we found no evidence for other alterations of either the mutant rhodopsin or the native rhodopsin from these animals.

Although the animals used for controls were SD rats, rhodopsin from RCS rats, not light damaged, was also examined and found to have identical sequence as the other wild type SD rats.

Discussion

The mass spectrometric analysis protocol developed for the mapping of integral membrane proteins and successfully applied previously to bacteriorhodopsin (BR) and bovine rhodopsin [28] proved equally applicable to rat rhodopsin. In spite of the small amount of protein (the samples contained approximately 175 mg or 5 nM of protein), we observed 90% of the entire sequence in a single experiment. The same posttranslational modifications were found in rat rhodopsin as had been reported for bovine rhodopsin [28]. Glycosylation was also found to be present with the same most abundant glycoform [28]. For rat rhodopsin, the Swiss-Prot gene sequence data reported a serine at 320 [45] as well as a possible threonine at this site (data not published). Our results show that rat rhodopsin contains a threonine at residue 320.

Our goal was to explore whether rat rhodopsin protein is damaged by extensive in vivo light exposure. Our results do not show evidence for any alterations or modifications in the primary sequence of the protein. Transgenic animals having a P23H mutation in rhodopsin are known to be extremely sensitive to light damage [42,43]. No light induced alterations in rhodopsin were found for these animals as well. This result was confirmed with the mass peaks of the base peak chromatograms and by sequencing individual peptide fragments. However, we did find unequivocal evidence that the mutated rhodopsin, as well as normal rhodopsin, is present in the rod outer segments from these animals. All rhodopsin fragments, except for the two single amino acid fragments and the glycosylated N terminal fragment were completely sequenced. The loss of masses corresponding to palmitates was also observed, strong evidence for the palmitylated state of the C terminal CNBr fragment.

It is well known that the C terminal region of light adapted rhodopsin is phosphorylated. Our results show extensive phosphorylation on the C terminal serines and threonines of rat rhodopsin. Previous in vivo experiments on mice that had received light flashes or continuous irradiation showed monophosphorylation at serine 338 and 334 respectively [37]. Hurley et al. reported multiple phosphorylations (mono to tri) of mouse rhodopsin isolated from intact retinae exposed to light in vitro [38]. In the experiments reported here, the rhodopsin was phosphorylated in vivo. The MS/MS data showed three main monophosphorylation sites and various diphosphorylation combinations at these sites. Triphosphorylation was also observed in various combinations of the three main monophosphorylation sites. We also obtained a very small intensity tetraphosphorylated peak based upon molecular weight measurement, but it was not confirmed by sequence analysis. This result agrees with the previous finding that extensive rhodopsin bleach causes multiple phosphorylations [38]. However, the extensive in vivo light damaging bleach, which leads to visual cell death, does not result in phosphorylation of >40% of the total amount of rhodopsin. Since the animals were sacrificed and the rhodopsin excised immediately after light damage, phosphatase activity should not be a major factor. However, the action of phosphatases in causing dephosphorylation cannot be ruled out. It was reported earlier that monophosphorylation may be present in fully dark-adapted retinas [38], and our results confirm that there may be a very small amount of rhodopsin phosphorylation (3-4%) in dark-adapted light damaged or control retinas. The MS/MS data show that the site of phosphorylation can be on any one of the three main monophosphorylation sites.

In conclusion, we have mapped rhodopsin peptides from control and light damaged rats by mass spectrometry and have confirmed that the amino acid at position 320 is threonine. Our results do not show any alterations in rhodopsin following exposure to levels of light that cause retinal damage, aside from the identification of multiphosphorylated states. However, after dark adaptation, no differences between the experimental and control groups were observed. Even using the light sensitive P23H transgenic animals, no modifications of the protein were observed. It cannot be ruled out that there is a form of the protein that is highly modified and therefore is not isolated through our sucrose flotation procedures, not cleaved in our fragmentation of the protein for mass spectrometry, or not eluted from our HPLC separation of the peptides. However, as the amount of protein isolated from the light damaged animals and the controls was equivalent and as the concentrations of the peptides appeared comparable, we conclude that the majority of the protein has not been altered. Our data therefore indicate that primary structural changes in rhodopsin are not a consequence of the well-known light damage effect that disrupts the organization of retinal photoreceptor cells.

Acknowledgements

We thank Drs. K. Schey and Jim Hurley for helpful discussions. Dr. M. LaVail generously supplied the P23H transgenic rats. Funding was provided by NIH grants EY-01959, EY-04939, and EY-08239; a private endowment to Dr. Organisciak from M. Petticrew and an unrestricted grant to MUSC from the Foundation for Prevention of Blindness. The mass spectrometry work was performed in the MUSC Mass Spectrometry Institutional Research Resource Facility.

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