Age-Related Protein Insolubilization in Lenses of CRYαAN101D and CRYαAWT Mice
To determine at what age there is change in the protein profiles in lenses of CRYαAN101D and CRYαAWT mice occurred, a comparative analysis of WS-proteins and WI-proteins from the lenses of the two types of mice of different ages was carried out (Fig. 1). The WS- and WI-proteins from lenses of different ages (1-, 3-, 4-, 5- and 7-months) were analyzed by SDS-PAGE. The WS-protein profiles from the lenses of the CRYαAN101D and CRYαAWT mice were almost identical until 3-months of age, except lens preparations from ages of 4-, 5- and 7-months of CRYαAN101D mice exhibited relatively greater levels of aggregated protein of Mr >30 kDa and higher relative to same-aged lenses from CRYαAWT mice (Lanes 4 and 5 in Figure 1B). Additionally, on quantification, the relatively increasing levels of WS-proteins showed age-related water insolubilization beginning at 4-months of age in the lenses of αAN101D mice (Table 1). Between 4- to 7-months of age, relatively about 5 to 10% higher proteins became insoluble in lenses of CRYαAN101D.To determine changes in individual crystallins due to their insolubilization, the WS-protein fraction from 7-month-old lenses was fractionated by a size-exclusion HPLC using a G-4000PWXL column (Tosoh Biosciences, fractionation range of protein with Mr’s between 1X104 to 1X107 Da). The comparative protein elution profiles at 280 nm of 7-month old lenses of αAN101D‑mice showed an increased protein in the void volume peak (representing WS-HMW proteins), and reduced β- and γ-crystallin peaks relative to lenses of CRYαAWT-mice (the differences shown in green in Figure 2A). The void volume peak in WS-protein fraction was also higher in the 7-month old lenses relative to 1-month old lenses of αAN101D‑mice (Results not shown), suggesting a relatively increased HMW protein aggregate formation with aging. On western blot analysis of the individual column fractions nos. 6 to 9 (constituting the void volume-HMW-protein peak) with an anti-His antibody, the levels of His-immunoreactive protein were higher in 7-month old CRYαAN101D lenses compared to the identical aged CRYαAWT lenses (Figure 2B). Additionally, because the immunoreactive peak in the WT lenses was in the fractions no. 8 and 9 whereas it was in the fractions no. 7 and 8 in the αAN101D lenses that suggested that the HMW proteins of αAN101D lenses showed a higher molecular weights relative to the HMW proteins from WT lenses. On quantification of Western blot images with Image J (Figure 2C), the intensity of the immunoreactive HMW proteins of αAN101D was about 20% greater relative to WT lenses. Together, the results suggested a greater aggregation with higher Mr of the HMW-proteins in CRYαAN101D lenses compared to the identical aged CRYαAWT lenses.
Identification Proteins Present in Water Insoluble-Urea Soluble (WI-US) - and Water Insoluble Urea Insoluble (WI-UI) Protein Fractions of Lenses of CRYαAWT and CRYαAN101D Mice
To Identify the insolubilized proteins in WTαA vs. αAN101D lenses, the WI-proteins from 5-month-old mice were further fractionated into WI-US- and WI-UI-protein fractions, and examined by SDS-PAGE (Figure 3) followed by their protein compositional analysis by mass spectrometry. SDS-PAGE analysis showed that both WI-US- and WI-UI-protein fractions from CRYαAN101D lenses contained greater levels of protein species including aggregated proteins (Mr > 30 kDa) [Identified as a, and c in Figure 3] relative to the same fractions from lenses of CRYαAWT mice (Identified as b and d in Figure 3). The mass spectrometric analysis was carried out at the following two levels: (i) In the first level analysis, determination of the total protein compositions in the WI-US- and WI-UI protein fractions of the two types of lenses (Supplemental Tables A [Comparative protein compositions of WI-US-fractions of CRYαAN101D and WTαA lenses], and Supplemental Table B [Comparative protein compositions of WI-UI-fractions of αAN101D and WTαA lenses]). (ii) In the second level analysis, the protein compositions of protein aggregates (Mr >30 kDa) in WI-US-fraction of αAN101D lenses (Identified as ‘a’ in Figure 3), and WI-US-protein fraction of WTαA lenses (Identified as ‘b’ in Figure 3) [Supplemental Table C]. Similarly, the compositions of protein aggregates (Mr >30 kDa) in WI-UI-fraction of αAN101D lenses (Identified as ‘c’ in Figure 3,), and WI-US-fraction of WTαA lenses (Identified as ‘d’ in Figure 3) were determined [Supplemental Table D]. The rationale of the two levels of analysis was to determine the relative proteins compositions due to the greater insolublization of proteins in CRYαAN101D lenses relative to CRYAAWT lenses (Figure 1, Table1). Our expectation was that the level 1 comparative examination would identify the total proteins that underwent insolubilization, and existed in the US- and UI-protein fractions, whereas the level 2 analysis would selectively identify those proteins that formed aggregates (Mr >30 kDa) in the US- and UI-fractions. The rationale was that the information would implicate potential roles of specific crystallins in the aggregation and therefore, in the cataractogenic mechanism.
(i) Comparative Protein Compositions in WI-US Fractions of Lenses from CRYαAN101D and CRYαAWT Mice
The proteins detected in the WI-US-protein fractions of CRYαAN101D lenses but were absent in the WT lenses are described in Supplemental Table A. Together, the results show that the WI-US fraction of CRYαAN101D lenses was enriched in several histones, which could be due to the lack of denucleation relative to WT lenses. Absence of Retinal dehydrogenase in transgenic lens fraction.
(ii) Comparative Protein Compositions of WI-UI-Fractions of Lenses from CRYαAN101D and CRYαAWT Mice
The proteins present in the WI-UI-protein fractions of CRYαAN101D lenses but were absent in WT lenses are described in Supplemental Table B. In summary, the results again show that the majority of histones that existed in CRYαAN101D lenses were absent in the WT lenses, which could be due to the lack of denucleation in the lenses of former mice. Also, specifically αB- and βB2-crystallin became insoluble as their levels were higher even in the WI-UI-fraction of lenses of CRYαAN101D relative to WT lenses.
(iii) Compositions of Aggregated Proteins (Mr >30 kDa) in WI-US- and WI-UI-Fractions of Lenses from CRYαAN101D and CRYαAWT Mice
As noted above, the purpose of the second level of mass spectrometric analysis was to elucidate the comparative compositions of aggregated proteins (Mr >30 kDa) in WI-US- and WI-UI-protein fractions of CRYαAN101D and CRYαAWT lenses [Supplemental Tables C and D]. On comparison, the major proteins present as aggregates (Mr > 30 kDa) in WI-US fraction of CRYαAN101D but absent in CRYαAWT were (Supplemental Table C): βB3- and γC-crystallins, collagen alpha-1(IV) chain and -alpha-2(IV) chain and nestin. In contrast, the exclusively present major proteins in WI-US fraction of CRYαAWT were: γC-, γD-, γE- γF-crystallins. The above list describes the selective proteins that were water insoluble-urea soluble and became the part of the complexes with Mr > 30 kDa in CRYαAN101D lenses. The greater abundance of αA-, and βB1-crystallins in the aggregated form suggested their potential involvement in the aggregation process along with βB3- and γC-crystallins.
On comparison of major proteins that existed in WI-UI protein fraction as > 30 kDa aggregates in CRYαAN101D not in the CRYαAWT included [Supplemental Table D]: γB-, γD- and γE-crystallins, and nestin. In the WI-UI fraction, the greater abundance of proteins in CRYαAN101D compared to CRYαAWT were: αA-crystallin and lens fiber intrinsic proteins. Together, the results showed that the proteins that remained urea insoluble and were possibly associated with the membrane of CRYαAN101D lenses included: γB-, γD- and γE-crystallins, and nestin (Nestin is an intermediate filament protein).
Increased Association of αAN101D with Lens Membrane in the Outer Cortical Fiber Cells relative WTαA in CRYYAAWT lenses
Our previous report  showed an increased levels and abnormal deposition of αAN101D within the outer cortical region in CRYαAN101D lenses compared CRYαAWT lenses. This suggested a relatively greater membrane binding of αAN101D, which was further investigated in experiments as described below.
(i) Immunohistochemical Analyses of Lenses from CRYαAN101D and CRYαAWT Mice
The purpose of the experiments was to determine relative levels of αAN101D and WTαA in the outer cortical regions of CRYαAN101D- vs. CRYαAWT lenses. This was examined by immunohistochemical analysis of 5-months old lenses of the two types of mice using anti-His monoclonal (for detection of WTaA and aAN101D [green fluorescence]) - and polyclonal anti-aquaporin 0 (for membrane detection [red fluorescence])-antibodies (Figure 4). The axial sections (at 10X magnification) showed an irregular and greater deposition of His-tagged aA (Green) in the lens outer cortex of CRYαAN101D mice (Shown by an arrow in Figure 4A) relative to CRYαAWT mice (Shown by an arrow in Figure 4B). Similarly, the equatorial sections (at 40X magnification) also exhibited a greater immunoreactive green fluorescence in the outer cortex of the CRYαAN101D lens relative to the CRYαAWT lens (shown by arrows in Figure 4C and D). Together, the results suggested the abnormally greater levels of association of αAN101D in the outer cortical regions, and potentially with the fiber cell membranes in the CRYαAN101D lenses relative to those of CRYαAWT lenses.
(ii) Relative Membrane-Association of WTαA- and αAN101D in Lenses of CRYαAN101D and CRYαAWT Mice
The rationale for the next experiment was that if the greater membrane-association of αA-N101D occurs in vivo in CRYαAN101D lenses compared to CRYαAWT lenses, the difference in their levels could also be determined in the purified membrane fractions by western blot analysis. The expectation was that following the step-wise membrane purification by using 8M urea (to dissociate non-covalently-bound membrane proteins), and by the final wash with 0.1N NaOH (to remove non-membranous extrinsic proteins) [30, 31], the purified membrane would show relative levels of membrane-association of αAN101D vs. WTαA in the two types of mice. To normalize the levels of the relative association during the membrane preparations, two lenses of 1-month-old and two lenses from 6-month old from CRYαAN101D and CRYαAWT mice were identically processed, using identical volumes of buffers at each steps during membrane purification (See Methods). Next, Western blot analysis using anti-His- and anti-aquaporin 0-antibodies were used to determine the relative levels of membrane-association of WTαA and αAN101D at different purification steps (Results not shown). To simplify the western blot results of fractions recovered during the sequential steps of membrane purification, only the results of immunoblots with anti-His antibody but not with anti-aquaporin-0 are shown in Figure 5. However, the western blot profiles with anti-aquaporin-0 were almost identical to anti-His antibody results. In Fig. 5, A, B, E and F show SDS-PAGE analysis followed by Coomassie blue-stained gels exhibiting relative levels of protein bands in preparations at different membrane purification steps in lenses at two different age groups (1 and 6 months). In Fig. 5, C and D (1-month old lenses) and G and H (6-months old lenses) corresponded to samples of A, B, E and F (Coomassie blue-stained gels), and show the Western blot results with anti-His antibody (green fluorescence) in the two different age groups (1 and 6 months). The levels of green fluorescence with His-tagged αA in lenses of 1-month old lenses (Figure 5, left panel: WTαA [C] and αAN101D [D]) and 6-month old lenses (Figure 5, right panel: WTαA [G] and αAN101D [G]) are shown. Additionally, in both left and right upper panels, the lanes 1, 2 and 3 show the WS-protein fractions recovered after first, second and third consecutive washes in buffer A to solubilize WS-proteins, respectively. Lanes 4 and 5 represent the urea soluble-protein fractions recovered during two consecutive washes of WI-protein pellet (containing membranes) with buffer B containing 8M urea, respectively. Lane 6 represents the 0.1N NaOH-solubilized proteins from membranes and the lane 7 from both 1- and 6-month old lenses (left and right panels) show the purified lens membrane preparations. Similarly, lanes 7 and 8 from 6-month old lenses (right panel) represent purified membrane preparation. Lane 9 of 6-month old lenses represents the crude lens WS-homogenate. The results show that the green fluorescence representing WTαA in CRYαAWT mice was entirely disappeared on urea solubilization in 1- and 6-month old lenses (lanes 1 to 5 in both left and right panels), whereas it was still present in these lenses until 0.1N NaOH wash (lane 6 in left and right panels). In contrast, the green fluorescence still existed in lane 6 of membranes from 1- and 6-month-old CRYαAN101D lenses. Together, the results suggest that αAN101D was tightly bound and at the higher levels to lens membrane of CRYαAN101D lenses relative to CRYαAWT lenses.
On Image J-quantification of the Western blots (Figure 5 I and J), the lanes 4 and 5 (urea soluble fractions) of 1-month old lenses showed higher levels (2.5X) of immunoreactivity with anti-His antibody in the CRYαAN101D lenses (shown in red) compared to those from CRYαAWT lenses (blue). Similarly in Figure 5J, among the lanes 4 and 5 containing same fractions from 6-month old lenses (as described in 1-month old lenses), the lane 5 showed a greater immunoreactive level of CRYαAN101D lenses (red) compared to CRYαAWT lenses (blue). Additionally, the lane 6 (representing membrane remaining after two urea washes, right panel) of 6-month CRYαAN101D lenses exhibited about 2X greater immunoreactivity than CRYαAWT lenses (Quantification results not shown). Together, the results show that relative to CRYαAWT, higher levels of CRYαAN101D were tightly associated with the lens membranes of 1- and 6- month old CRYαAN101D mice.
(iii) Relative Membrane-Binding of Alexa 350-Labeled Recombinant WTaA- and aA-N101D Crystallins To examine whether αAN101D show a greater binding affinity to the lens membrane relative to WTaA-crystallin, the binding of the two recombinant proteins to purified lens membrane was determined. The recombinant WTaA- and aAN101D proteins were labeled with Alexa 350 using a protein labeling kit by the procedure described by the manufacturer (Molecular Probes, Thermo fisher Scientific). The two labeled-proteins were purified by a size-exclusion HPLC column and were analyzed by SDS-PAGE. Figure 6A shows the Coomassie blue-stained WT aA (lane 1), aAN101D protein (lane 2), and the purified lens membrane from non-transgenic C57 mice (lane 3). The Figure 6B shows the images of the two Alexa 350-labeled proteins under a UV trans-illuminator [Lane 1: Images of Alexa 350-labeled WTaA, and lane 2: Alexa 350-labeled aAN101D). During the binding assay, the purified lens membrane (containing 2.5 mg protein; isolated from 1 to 3-month old non-transgenic C57BL mice) was incubated with increasing but identical concentrations of either Alexa-labelled WT aA- or aAN101D proteins at 37οC for 6 h (See details in Methods). A relatively higher levels (> 1.5X) of binding of aAN101D protein relative to WTaA protein with membrane preparation was observed (Figure 6C). The values reported are the average of triplicate assays.
(iv) Immunogold-Labeling for Relative Localization of αA-WT and αAN101D in Lens Membranes of CRYαAN101D and CRYαAWT Mice
To ascertain the relative levels association αAN101D vs. WTαA to the lens membrane in vivo, the immunogold-labeling experiment was carried out (See details in Methods). (A) and (B) in Figure 7 show lens membranes from CRYαAN101D and CRYαAWT mice at 500 nm magnification, and (C) and (D) from these lenses at 100 nm magnification, respectively. The bigger gold particles (25 nm, red arrows) the smaller gold particles (10 nm, yellow arrows) represented the aquaporin-0 and the His-tagged αAN101D and WTαA, respectively. As shown in the representative images in (A) to (D), the 25 nm gold particles (representing aquaporin-0, identified by red arrows) were bound to membranes. On counting the membrane-associated 10 nm particles (representing His-tagged αAN101D and WTαA), almost the same numbers of the particle were found to be associated with membranes of both CRYαAN101D and CRYαAWT lenses, suggesting that the His-tagged αAN101D and WTαA were bound to the membranes of the two types of lenses. Our previous study  showed that αAN101D protein constituted about 14% and 14.2% of the total αA- crystallin in the WS- and WI-proteins, respectively in the lenses of CRYαAN101D mice. Therefore, an argument can be made that although an almost equal number of 10 nm and 25 nm particles were associated with membranes of the two type of lenses, a higher number of gold particle representing αAN101D relative to WTαA were associated with the membrane.
Another interesting observation was that the membranes of CRYαAN101D lenses were about 2X more swollen relative to those of CRYαAWT lenses [Figure 7, compare (A) to (B) and (C) to (D)]. The width of the membrane was quantified using Image J as shown in Figure 7E. The swelling could represent water intake within the lens cells due to the potential ionic imbalance in the CRYαAN101D lenses compared to CRYαAWT lenses. Such a possibility of ionic imbalance was further determined as described below.
Na, K-ATPase and Ca2+ Levels in Cultured Epithelial Cells from Lenses of CRYαAN101D and CRYαAWT Mice
Sodium-potassium-adenosine triphosphatase (Na, K-ATPase) has been recognized for its role in regulating electrolyte concentrations in the lens, and the electrolyte balance is vital to lens transparency [35, 36]. In addition, calcium has been reported to control both sodium and potassium permeability through lens membranes . In our previous study , we showed that the expression of Na,K-ATPase at the protein level was drastically reduced in CRYαAN101D lenses relative to CRYαAWT lenses. Next, the levels of Na, K-ATPase mRNA, and Ca2+ levels were determined in cultured epithelial cells from lenses of CRYαAN101D and CRYαAWT mice. Both (A) and (B) in Figure 8 show intracellular Ca2+ levels in the presence of calcium orange in cultured epithelial cells from CRYαAN101D and CRYαAWT, respectively. Only a few CRYαAN101D epithelial cells showed the Ca2+ uptake, which was possibly due to our previous finding that the lens cells contained only about 14% of αAN101D mutant protein . In this experiment, 100 cells from the cultures of two types of lenses were counted. On quantification by Image J, the number of cells exhibiting calcium orange uptake were 1.5X greater in cells of CRYαAN101D lenses relative to cells from CRYαAWT lenses (Figure 8B). On the determination of levels of mRNA of Na, K-ATPase in these cells, its level was 75% lower in the CRYαAN101D lens cells than CRYαAWT lens cells (Figure 8C).