SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

The ultraviolet (UV) absorption of various sections of the human lens was studied and compared with protein expression paralleling differential UV absorbance in anterior and posterior lenticular tissue. The UV absorbance of serial lens cryostat sections (60 μm) and that of lens capsules was determined using a Shimadzu scanning spectrophotometer, and the absorption coefficients were calculated. Two-dimensional gel electrophoresis was performed using two pooled lenticular protein extracts (anterior and posterior sections). Protein spots were quantified and significantly different spots were identified by mass spectrometry following in-gel digestion with trypsin and chymotrypsin. The UV-C and UV-B absorption of the human lens increased toward the posterior parts of the lens. The anterior and posterior lens capsules also effectively absorbed UV radiation. Levels of molecular chaperone proteins Beta-crystallin B2 (UniProtKB ID:P43320), A3 (UniProtKB ID:P05813) and of glyceraldehyde 3-phosphate dehydrogenase (UniProtKB ID:P04406) were significantly higher in the anterior part of the lens, whereas lens proteins Beta-crystallin B1 (UniProtKB ID:P53674) and Alpha-crystallin A chain (UniProtKB ID:P02489) were higher in the posterior sections. These results provide evidence that differential UV absorption in the anterior and posterior lens is accompanied by differential protein expression.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Ultraviolet (UV) radiation poses an increasing risk on the tissues and organs exposed to radiation. In the human eye, the cornea absorbs most of the UV radiation [1], but still significant radiation may reach the lens. It is well established that UV radiation, especially within the range of UV-B (280–330 nm), may cause ocular damage [2, 3]. Exposure to near UV light (315–400 nm) has been reported to lead to pathological changes in the lens of albino mice and gray squirrels [4, 5]. Several studies provided evidence for the relationship between the risk of cataract and sun exposure [2, 6-9]. Brown type of human cataracts develops much more frequently in outdoor-active individuals than in indoor workers [9] and an increased risk of posterior subcapsular cataract is associated with high exposure to UV-B radiation [6].

The primate lens absorbs most of the UV-A (330–400 nm) and UV-B (280–330 nm) radiation [10]. Accordingly, it is thought that under normal circumstances no considerable amounts of UV radiation reach the retina. Therefore, it is of critical importance to determine to what degree various sections of the lens and its capsule contribute to the capacity of the lens to absorb UV radiation. An earlier study has shown that the various layers of the cornea are able to absorb UV radiation to different extent [1]. There are only few studies concerning the absorption properties of the different layers of the lens. Gaillard et al. [11] found, that in primates, the posterior layers of the lens show higher UV absorbance than the anterior ones in the 300–440 nm range. Another study by Dillon et al. [12] showed that the nucleus of an old human lens reveals higher absorbance than the outer cortex. Moreover, no UV absorption data are available on the lens capsules.

The most abundant known low–molecular-weight UV-filter compounds in the human lens are kynurenine, 3-hydroxykynurenine glucoside (3OHKG), and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside [13]. The UV spectra of these molecules show higher absorbance in the UV-B and UV-C ranges than in the UV-A range. Results suggest that these tryptophan-derived UV filters are responsible for UV radiation absorbance in the lens. Levels of these UV filters and protein-bound kynurenine and 3-hydroxykynurenine (3OHKyn) are higher in the nucleus than in the cortex [14]. Accordingly, the nuclear concentration of free UV filters 3OHKG and 3OHKyn has been shown to be lower than that in the cortex [15]. Apart from these low-molecular-weight compounds proteins may significantly contribute to UV absorption capacity.

The human lens has the highest protein content within the body and all proteins are absorbing in the UV range [16]. Key proteins, i.e., crystallins of the lens have earlier been described in detail as major constituents and recently identified by mass spectrometry, too [17]. These specific lenticular proteins are main determinants for optical properties including UV absorbance. To the best of our knowledge, however, the presence of these proteins was not localized to anterior or posterior parts of the lens.

It was therefore the aim of the current study to determine the UV absorption of various parts of the lens and the lens capsules and to carry out a gel-based proteomic study of human anterior and posterior lenticular parts. And indeed, anterior and posterior sections showed differential protein expressional patterns that were paralleling differential UV absorbance. Our findings on differential crystallin expressions in the anterior and posterior sections are in agreement with previous work on the role of lenticular proteins for UV absorption [18, 19].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Cryostat sectioning

Human lenses (n = 25) were obtained from eyes with a post-mortem delay of 6 h. Most of the available lenses were between the age of 40 and 60 years and this age group was split in two: lenses taken from 40–49 (n = 10) and from 50–59 (n = 9) years old eyes were used for UV-absorption measurements. The rest of the lenses (n = 6) were used for proteomic studies.

The human lenses were used with the approval of the Human Ethics Committee of University of Szeged, Albert Szent-Györgyi Clinical Centre and the study was conducted in accordance with the provisions of Declaration of Helsinki for experimentation involving human tissue. The eyeballs were transported and stored in a humid chamber at 4°C until the lenses were removed. Lenses were stored at −70°C until used and did not reveal any cataract formation. To determine the absorbance of the various sections of the lens, 60 μm thick consecutive cryostat sections were cut at −18°C. The day before sectioning, the lenses were placed into cold 30% sucrose in PBS and stored at +4°C. The absorbance of 30% sucrose solution was determined (60 μm thick layer) and was found negligible. Before sectioning, the anterior and posterior lens capsules were removed and stored in physiological saline separately. During cryostat sectioning, the anterior 200 μm thick portion of the lens was discarded, so the first section was taken from 200 μm anteroposteriorly (Fig. 1). The next two sections were not used and the second sample was taken posterior to these. Accordingly, we had two 60 μm thick samples from the anterior cortex. Posterior to that, every seventh section was kept for measurement. Thus, five samples were taken from the nucleus, and two samples from the posterior cortex with a 120 μm thick discarded portion between them. Altogether, nine sections were taken for UV measurements in an anteroposterior order. Figure 1 shows the position of the sections in the lens and Table 1 summarizes the calculated anteroposterior depth of sections used for UV absorbance measurements. To avoid light scattering, the lens samples and the capsules were placed between two quartz glass plates with a drop of isotonic saline on both sides of the specimen. A 60 μm thick spacing ring was placed between the plates to prevent compression damage to the samples.

Table 1. The calculated anteroposterior position of the sections processed for UV measurement within the lens. Average thickness of the whole lens was approximately 3.95 mm
Sample123456789
Anteroposterior depth (mm)0.200.380.801.221.642.062.483.563.74
image

Figure 1. The position of the different sample layers within the lens. The first two samples [1, 2] were taken from the anterior cortex, the next five [3-7] from the nucleus and the last two samples [8, 9] derived from the posterior cortex. Layers 1 and 2 as well as 8 and 9 were used for proteomics studies.

Download figure to PowerPoint

UV absorbance spectrophotometric measurements

UV absorbance of samples was measured by a scanning spectrophotometer (model UV-2101PC UV-VIS; Shimadzu, Kyoto, Japan). This model contains two spectral lamps (halogen lamp for 900–340 nm, and deuterium lamp for 340–190 nm wavelength ranges), a double monochromator to select the proper wavelengths, measuring and referencing beam paths, and a photomultiplier to detect and amplify the measuring and reference light intensities. On the reference side, only a drop of isotonic saline was placed between the quartz plates. The spectrophotometer was connected to a computer and the absorption spectrum was analyzed using the software of the spectrophotometer. The absorbance of the central portions of the lens sections was determined using an aperture with 2 mm diameter thus only the representative part of lens was measured (anterior and posterior cortex and nucleus), no matter, whether the peripheral parts of the section involved other parts of the lens (e.g. cortex in case of sections of the nucleus, See Fig. 1 for details). The absorbance and the absorption coefficient of the samples were calculated as in our earlier report [1]. The investigated wavelength range was 200–400 nm. The absorbance measurement range of our device was between 0 and 5 as the recorded absorbance values were often found to be greater than 4 at wavelengths below 240 nm. Therefore, the accuracy of our measurements in the studied wide measuring range did not make possible to detect minute changes of absorbance as observed by Gaillard et al. around 360 nm wavelength [11]. As the recorded results often approached or reached the upper instrument rating, therefore we present the absorbance data only in 240–400 nm range in our calculations.

Following the absorbance measurements, cryostat cross sections were cut from the anterior and posterior capsules. Absorbance was determined after trypsin treatment only as the repeated measurement procedure destroyed the thin samples. Therefore, anterior capsules with the same characteristics (age and thickness) were used as positive controls.

Proteomic studies

Two-Dimensional Gel Electrophoresis (2DE). Cryostat sections were cut from six lenses as described above and the anterior lenticular pool of sections consisted of sections 1 and 2, the posterior lenticular pool consisted of sections 8 and 9. These pools were stored at −80°C until gel-based proteomics studies. 2DE was performed essentially as reported previously [20-22].

Quantification of protein levels. Excess dye from 2DE gels were removed by washing with distilled water and gels were scanned with an Image Scanner (Amersham Pharmacia Biotech). Protein spots from each gel (six gels per group, n = 12) were outlined (first automatically and then manually) and quantified using the ProteomweaverTM software (Bio-Rad, CA, USA). The percentage of the volume of the spots representing a certain protein was determined in comparison with the total proteins present in the 2DE gel [23]

Analysis of peptides by Nano-LC-ESI-MS/MS (High-capacity ion trap, HCT). Fourteen spots which showed different levels between group 1 (anterior pool) and group 2 (posterior pool) were manually excised and placed into 1.5 mL Protein LoBind Eppendorf tubes. In-gel digestion and sample preparation for HCT analysis was performed as described before [23]. The extracted peptides were pooled for Nano-LC-ESI-CID/ETD-MS/MS analysis as described in a previous article [24]. Protein identification and modification information returned from MASCOT were manually inspected and filtered to obtain confirmed protein identification and modification lists of CID MS/MS and ETD MS/ MS. Posttranslational modification searches were done using ModiroTM v1.1 software (Protagen AG, Germany). Protein identification and modification information returned were manually inspected and filtered to obtain confirmed protein identification and modification lists. [24].

Verification of protein expression levels as evaluated by ProteomeweaverTM by in-gel ninhydrin assay. Each protein spot was cut out and put into a 0.5 mL Protein LoBind tube (Eppendorf, Hamburg, Germany). Spots were initially washed twice with ACN: de-ionized water (1:1, v/v) for 20 min. Subsequently 100 μL of 100% ACN was added to each tube to cover the gel piece completely and incubated for at least 2 min. After the removal of ACN, gel pieces were dried completely in a SpeedVac Concentrator 5301. The dried gel pieces were reswollen with 60 μL of 2 ng/μL proteinase K (Promega, Madison, WI, USA) buffered in 1 mM HCl. Gel pieces were incubated for longer than 4 h at 37°C. Supernatants were transferred to new 0.5 mL tubes, and gel pieces were extracted by 80 μL of 1% formic acid (Fluka, Steinheim, Germany) under sonication for 30 min. Following this extraction step, peptides were eluted from the gel pieces by 20% ACN under sonication for 30 min and subsequently by 40% ACN for 30 min. The individual extractions were pooled into the 0.5 mL LoBind tubes containing the supernatant including proteinase K. The volume was reduced to a final volume <10 μL in a SpeedVac concentrator and undergoing acid hydrolysis and ninhydrin reaction. Ninhydrin reaction was carried out as follows: Samples were hydrolyzed in 0.5 mL Eppendorf Protein LoBind tubes with 150 μL of 6 N hydrochloric acid solution (Fluka) at 99°C for 24 h. Hydrolysates were spun down and evaporated in an SpeedVac concentrator (Eppendorf). The dried material was redissolved in 20 μL of distilled water, vortexed and centrifuged. Ninhydrin working solution was prepared as referenced [25]: 200 mg of ninhydrin reagent (Sigma Chemical Co., Steinheim, Germany) was dissolved in a mixture of 7.5 mL of ethylene glycol (Sigma Chemical Co.) and 2.5 mL of 4 M acetate buffer and 250 μL of the stannous chloride (Sigma Chemical Co.) solution (add 50 mg of SnCl2 to 500 μL of ethylene glycol) were added with stirring. The reagent should be pale red in color. A quantity of 100 μL of the final mixture was transferred to a flat bottom NUNC MaxiSorp microtiter 96-well plate (Thermo scientific, Langenselbold, Germany) and the mixture was kept in an incubator (MELAG OHG Medizintechnik, Berlin) at 100°C for 20 min. Absorption was measured on an iMark microplate reader (Bio-Rad) at 570 nm. Measurement of all the samples was performed within 10 min [25].

Statistical analysis

Statistical analysis to reveal between-group differences was performed by paired Student's t-test. Bonferroni-Holm and LSD correction was applied for correction of multiple testing. In all proteomic studies, a probability level of P < 0.01 was considered statistically significant. All calculations were performed using SPSS version 14.0 (SPSS Inc., Chicago, IL).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

The highest absorption levels of the samples were detected in the UV-B and UV-C range. Figures 2A–C show the averaged absorption coefficients of 10 samples in the first, and nine samples in the second age group as function of wavelength in the range of 240–400 nm.

image

Figure 2. (A) Averaged absorption coefficients of the nine investigated layers as a function of wavelength in the age group of 40–49 years (n = 10). (B) Enlarged view of the curves shown in A in the 340–400 nm wavelength range. Note the fine elevated UV absorption at 360 nm. (C) Averaged absorption coefficients of the nine investigated layers as a function of wavelength in the age group of 50–59 years (n = 9).

Download figure to PowerPoint

In the UV-A range, a slow increase of absorption coefficient can be seen, which continues in the UV-B range too. At 360 nm, a mild and transient increase of absorption coefficient is observed, which can be best seen when the range of absorption coefficient is scaled only up to one unit (Fig. 2B). From about 310 nm, the absorption coefficient steeply increases and reaches a maximum at 280 nm (end of UV-B range). From this wavelength to 250 nm the absorption coefficient decreases and then increases again. The samples, which were taken from more posterior parts of the lens, show the same characteristics, but the absorption coefficients of the posterior layers are higher. For example, at 280 nm, the absorption coefficient of the eighth sample is approximately five times greater than that of the first one.

Absorption coefficients of the anterior (n = 14) and posterior (n = 13) lens capsules, and the average of these coefficients were calculated too (Fig. 3), as function of wavelength.

image

Figure 3. Averaged absorption coefficients of the anterior (CA) and posterior (CP) lens capsules as function of wavelength. Posterior capsules have higher absorption coefficients relative to anterior capsules at the wavelength of 280 nm. Note the minor age difference in the averaged absorption coefficient values of the posterior capsules at wavelengths 250–280 nm.

Download figure to PowerPoint

It appears that the absorption of the capsules shows the same characteristics as the various layers of the lens and absorption coefficient of the lens capsules have approximately the same value as the anteriormost layer of the lens (Fig. 2A and B). Absorption is more effective in UV-B and UV-C ranges than in UV-A range, with absorption maximum at 280 nm. Moreover, it is evident that the posterior lens capsules have greater absorption coefficients than the anterior capsules in each age group but this difference was not significant. The thickness of the anterior lens capsule changes with age while that of the posterior capsule is approximately constant [26]. This was confirmed by our measurements too. Thickness of the anterior lens capsules increased with age, from 20.92 μm (age of 42 years) to 24.74 μm (age of 57 years), while that of the posterior capsules remained relatively constant between 8.22 and 10 μm.

The absorbance of the anterior lens capsule is given together by the absorbance of the capsule and that of the epithelial lining. To determine the absorbance of the epithelium, the absorbances of separate lens capsules with or without epithelium (after trypsin treatment), but possessing identical properties (age and thickness) were measured. Then from the difference of the two values the absorbance and the absorption coefficient of the epithelium were determined. Figure 4 shows the absorbance of a 55 years old anterior lens capsule with epithelium and that of a 62 years old anterior capsule without epithelium. It can be seen that the two lines differ only at wavelengths below 300 nm. The difference of the two values proposes the absorbance of the epithelium. It appears that the epithelium does not contribute considerably to absorbance.

image

Figure 4. Absorbance of the anterior lens capsules with and without epithelium. The continuous line shows the calculated absorbance of the epithelium (EP). The anterior capsules (CA) had comparable physical properties.

Download figure to PowerPoint

To determine which proteins may underlie anterior–posterior differences of UV absorption, a gel-based proteomics study was carried out. 2D gel electrophoresis revealed a large series of spots (n = 146). A representative two-dimensional gel identifying proteins with differential levels in group 1 (samples 1 and 2) and group 2 (samples 8 and 9) along with their corresponding UniProtKB numbers is shown in Fig. 5.

image

Figure 5. Picture A and B show the master gels of group A and group B, respectively, with the significantly changed 9 spot volumes from eye lens samples. UniProtKB accession numbers are provided. Several spots indicate the presence of identical proteins Beta-crystallin B1 and glyceraldehyde-3 phosphate dehydrogenase.

Download figure to PowerPoint

Quantification results of nine proteins with significantly different levels between group 1 and 2 are listed in Table 2. Identification of these nine protein spots along with their matched peptide numbers, sequence coverage and identification by Mascot software including ion scores/mass errors and MS/MS peptides determined are listed in Table S1. MASCOT software is one of the most universal algorithm for mass spectrum analysis and it can also predict or identify posttranslational modifications (PTMs) or other modifications such as amino acid conflicts. Complementary to the MASCOT search, ModiroTM search was also performed and as shown in Table S2 all five individual proteins were identified with high significance and ion scores. Table S3 shows the sequence coverage of all five individual proteins from different enzyme digestions, conditions, and different search algorithms. By combining results from different enzymes and bioinformatic algorithms, high sequence coverage (maximum 90.83%) was obtained. All peptides were identified from trypsin, chymotrypsin digestion, proteases widely used for mass spectrometric analysis. Verification of protein expression levels from each protein spot of 2DE gels was performed using in-gel ninhydrin assay. Table 2 shows the results of the two different quantification methods providing means, SD, and P-values. Human glyceraldehyde 3-phosphate dehydrogenase, Beta-crystallin B2, and Beta-crystallin A3 levels were higher in the anterior lenticular pool than in the posterior. Levels of the Human Alpha-crystallin A chain and that of the Human Beta-crystallin B1 were higher in the posterior pool. The representative MS/MS spectra of Beta-crystallin B1 and Human glyceraldehyde 3-phosphate dehydrogenase and identified PTMs with a-, b-, y-, B-Pi,and y-Pi ion series that were identified either with MASCOT or ModiroTM are shown in Figure S2–S9.

Table 2. Proteins present in significantly different levels in the lens. The P-values of the two analyses are corresponding with each other
Spots with differentially expressed proteinsVerification of protein expression levels by in-gel ninhydrin assay
1.-2.8.-9.P-valueProteinAccession No.a1.-2.8.-9.P-value
meansdmeansdmeansdmeansd
0.2990.1150.0890.0570.005G3P_HUMAN Glyceraldehyd-3-phosphate dehydrogenaseP044060.0650.0150.0210.0080.007
0.4230.1731.0780.3640.003CRYAA_HUMAN Alpha-crystallin A chainP024890.0960.0190.2110.0270.001
0.5940.2391.5220.5030.002CRBB1_HUMAN Beta-crystallin B1P536740.1470.0170.3980.0680.001
0.4720.3811.6070.5920.003CRBB1_HUMAN Beta-crystallin B1P536740.1110.0090.3970.0150.001
0.1970.1821.1040.3730.001CRBB1_HUMAN Beta-crystallin B1P536740.0400.0140.2690.0320.001
1.0790.2410.4620.1670.000CRBB2_HUMAN Beta-crystallin B2P433200.2420.0270.1010.0210.001
1.0950.3430.4720.1040.004CRBA1_HUMAN Beta-crystallin A3P058130.2470.0200.1270.0140.001

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Herein, UV absorbance differences at various anteroposterior sections of the human lens and that of the anterior and posterior lens capsules in the range of 240–400 nm were studied. It was observed that the posterior layers of the lens reveal higher absorption coefficients than the anterior layers at wavelengths shorter than 310 nm (data in Fig. 2). These results correspond to data reported in literature. In addition, it was observed that the posterior lens capsules show higher absorption in the UV-B and UV-C range than the anterior capsules (Fig. 3). Our data suggest that the epithelium of the anterior capsule does not exhibit significant UV absorbance (Fig. 4). It is noted, however, that we found only minor differences between the two age groups (40–49 years and 50–59 years) both at the level of individual parts of the lens or that of cumulative parameters.

To demonstrate the UV-filtering effect of the different layers of the lens, absorbance of the whole lens together with the lens capsules was calculated from the data presented above. First, the absorbance of the anterior capsule was computed, subsequently the absorbance of the anterior cortex was calculated and added to that of the anterior capsule. Analogously, absorbances of the nucleus, posterior cortex, and posterior capsule were added to that of the anterior layers, thus calculating the absorbance of the whole lens. Figure 6 shows cumulative absorbance data as function of wavelength in the age group of 40–49 years. It is evident that the nucleus mainly contributes to the whole absorbance by its thickness.

image

Figure 6. Cumulative absorbance of the whole lens. Absorbances of the various structural layers 1–9 were added together.

Download figure to PowerPoint

Similarly, transmission of the whole lens was calculated. Figure 7 shows that at wavelengths shorter than 290 nm the anterior capsule absorbs about 40% of the UV radiation and the transmission is reduced to approximately 0% by the rest of the lens structures. From the practical point of view, the transmission parameters of the investigated part of ocular system are also important. Accordingly, the transmission of the capsule, that of the whole lens and the complete lens capsule system have also been calculated (Fig. 7). Figure 7A shows that at wavelengths shorter than 290 nm the anterior capsule transmits about 60% of the UV radiation while the transmission is reduced to approximately 0% by the rest of the lens structures (Fig. 7B). To verify our measured and derived values, we determined the transmission of a whole lens using a special measuring arrangement. Figure 7C shows the transmission curve of a 51-year-old lens. The most important wavelength range was magnified (Fig. 7D) to compare the tendencies of the transmission curves concerning the whole lens obtained by direct measurement and indirect calculation. It can be seen clearly that the tendencies are similar, with a clearly observable displacement/difference of the curves which are due to the reflection and light scattering effects on surfaces of the investigated lens sections. While these effects are negligible in the case of a single lens layer (60 μm) measurement, they are added up when the total lens transmission is calculated from the values of several lens sections. However, we were not able to remove these scattering effects in our transmission calculations for the whole lens.

image

Figure 7. (A) Calculated transmission values of anterior capsule and that of the anterior capsule and the anterior cortex together (50–59 years age group). (B) Calculated transmission values of anterior capsule, anterior cortex and nucleus together, that of the anterior capsule, anterior cortex, nucleus and posterior cortex together and that of the whole lens with lens capsules. Note the considerable transmission loss of UV in the anterior parts of the lens. (C) Measured transmission of a 51-year-old whole human lens. (D) Comparison of the measured and calculated transmission values for the whole lens in the 360–450 nm wavelength range (data are obtained and shown along an enlarged scale from the intact whole lens shown in C and from calculated transmission values shown in B). Note that the actual values of the measured and calculated cut-off wavelengths are quite similar (376 vs 390 nm), although the curves show considerable displacement at greater transmission values, such as from 0.5%.

Download figure to PowerPoint

To understand the rate of the absorption changes within the lens, the values of the absorption coefficients of the various layers at 280 nm are shown as function of anteroposterior depth in Figure. S1 (see supplementary data).

Studying protein expression differences between the anterior and posterior lenticular pools revealed that levels of human Beta-crystallin B2 and A3 and that of glyceraldehyde 3-phosphate dehydrogenase were significantly higher in the anterior cortex of the lens whereas levels of Alpha-crystallin A chain and Beta-crystallin B1 were higher in the posterior segments. Crystallins are major lenticular proteins and serve a series of functions including chaperoning, contribution to transparency and refractive index, and determine lens structure and physicochemical properties. The alpha-crystallins are thought to play the roles of molecular chaperones and are members of the small heat shock protein family, moreover, they are supposedly associated with a number of neurological disorders. The beta/gamma-crystallin superfamily possesses well-defined N- and C-terminal extensions, being responsible for their distinct biophysical and biochemical properties. Mutations in the beta/gamma-crystallin genes may lead to opacification of the lens.

The individual crystallins may play distinct roles under UV irradiation: Alpha-crystallin, a major constituent of the lens inhibits UV-light-induced aggregation of other lens proteins [27] and is thought to be a major target for UV irradiation due to its higher degree of unfolding [28]. Earlier studies by Liang et al. [18, 19] have shown that tryptophan residues of alpha-crystallin are more exposed to UV radiation due to their greater degree of protein unfolding than those of beta or gamma-crystallins, and the unfolding of alpha-crystallin further increases with aging. Accordingly, as tryptophan residues are mainly responsible for the peak UV absorbance of the lens at 280 nm, alpha-crystallin is suggested to play a major role in UV absorption compared with beta or gamma-crystallins. Therefore, it is not surprising that increased amounts of alpha-crystallin in the posterior cortex may induce greater UV absorption than anterior parts enriched in beta-crystallin isoforms, which display lesser ability to absorb UV radiation. Moreover, sites of attachment of UV filters could be identified and linked to betaB1-crystallin [29]. Gamma-crystallin D is an essential lens protein and exposure to UV-C is thought to perturb protein structure and probably leading to aggregation [30] and a gamma-crystallin fold may have been evolved to protect tryptophan from UV photodamage [31].

These crystallins may present the molecular basis for differences in the anterior and posterior UV absorbance because the higher degree of unfolding of Alpha-crystalline A and Beta-crystalline B1 chains may render the posterior part of the lens a more UV absorbing structure. The manifold increase of glyceraldehyde-3-phosphate dehydrogenase in the anterior section pool may be due to and reflect differences in carbohydrate metabolism within the lens. Interestingly, this enzyme was described as a target of UV-B [32].

It remains open whether differential crystallin localization underlies or reflects different optical or biological functions of the lens.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

We acknowledge the contribution by the Verein zur Durchführung der wissenschaftlichen Forschung auf dem Gebiet der Neonatologie und Kinderintensivmedizin “Unser Kind”. The authors are grateful for the support of the Hungarian Scientific Research Fund (OTKA, grant nos. T/F 043371 and K 67818). This work was partially supported by the Office for National Research and Technology (NKTH, project number CNK 78549).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
php12063-sup-0001-TableS1.docWord document492KTable S1. Identification of lens protein spots by Mascot software including ion scores/mass errors and MS/MS peptides.
php12063-sup-0002-TableS2.docWord document67KTable S2. Modifications of identified lens proteins (Modiro)
php12063-sup-0003-TableS3.xlsxapplication/msexcel28KTable S3. Sequence coverage of all five individual proteins from different enzyme digestions, conditions and different search algorithms
php12063-sup-0004-FigS1.tifimage/tif5283KFigure S1. The values of the absorption coefficients of the various layers at 280 nm are shown as function of anteroposterior depth.
php12063-sup-0005-FigS2-S10.pptapplication/mspowerpoint762KFigure S2–S9. The representative MS/MS spectra of beta-crystallin B1 and human glyceraldehyde 3-phosphate dehydrogenase and identified PTMs with a-, b-, y-, B-Pi, and y-Pi ion series are shown.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.