Age-Related Protein Insolubilization in Lenses of CRYAAN101D and CRYAAWT Mice
The WS- and WI-protein fractions were isolated from two lenses of CRYAAN101D and CRYAAWT mice of ages of 1-, 3-, 4-, 5- and 7-months, and their protein profiles were compared by SDS-PAGE analysis. To normalize comparison among WS-and WI-protein profiles of the lenses of the mice of different ages, the lenses from each age mice were identically processed using identical volumes of buffers to recover their WS- and WI-proteins. Further, an equal volume of WS- or WI-proteins preparations from lenses of each ages of the two types of mice were used during the SDS-PAGE analysis. These normalizing steps allowed the comparative determination of profiles of age-related changes in the WS- and WI-proteins of the lenses. The comparative WS-protein profiles from the lenses of the CRYAAN101D (identified as transgenic in Fig.1) and CRYAAWT (identified as WT in Fig. 1) mice were almost identical up to 3-months of age. However, the WI-protein preparations from CRYAAN101D mice of 4-, 5- and 7-months showed relatively higher levels of proteins, which included levels of crystallins (Mr between 20-35 kDa) and their aggregated species (Mr > 45 kDa) in the αAN101D lenses relative to wild-type lenses beginning at 4- months of age (Lanes 4 and 5 in Fig 1B). This suggested that the increasing levels of WS-proteins showed age-related water-insolubilization beginning at 4-months of age in the lenses of αAN101D mice (see Table in Figure 1). Between 4- to 7-months of age, about 5 to 10% higher proteins became water insoluble in lenses of CRYAAN101D relative to lenses of WT mice. To determine changes in individual crystallins due to their insolublization, 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 kDa). The comparative protein elution profiles at 280 nm of 7-month old lenses of CRYAAWT and CRYAAN101D mice showed an increased protein in the void volume peak (representing WS-HMW proteins), and reduced β- and γ-crystallin peaks in the latter (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 CRYAAN101D mice (Results not shown), suggesting an 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 His-immunoreactive protein levels were higher in 7-month old CRYAAN101D lenses compared to the identical aged CRYAAWT lenses (Figure 2B). Additionally, 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, suggesting that the HMW proteins of αAN101D lenses had a higher molecular weight relative to the HMW proteins from WTαA lenses. On quantification with Image J, the intensity of the immunoreactive HMW proteins of αAN101D was about 20% greater relative to WTαA-lenses. Together, the results suggested a greater aggregation with higher Mr of the HMW-protein fraction in αAN101D lenses relative to WTαA lenses.
Identification Proteins Present in Water Insoluble-Urea Soluble (WI-US) - and Water Insoluble Urea Insoluble (WI-UI) Protein Fractions of Lenses of CRYAAWT and CRYAAN101D 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, examined by SDS-PAGE (Figure 3), which was followed by mass spectrometric analysis to determine their protein compositions. SDS-PAGE analysis showed that both WI-US- and WI-UI-fractions from CRYAAN101D lenses contained greater levels of proteins including aggregated proteins (Mr > 30 kDa) [Identified as a and c in Figure 3] relative to the same fractions from lenses of CRYAAWT 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 (A) Tables 1 [Comparative protein compositions of WI-US-fractions of αAN101D and WTαA lenses], and Supplemental (B) Table 2 [Comparative protein compositions of WI-UI-fractions of αAN101D and WTαA lenses]). (ii) In the second level analysis, the protein compositions of aggregates (Mr >30 kDa) in WI-US-protein 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 (C) Table 3]. 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' Figure 3) were determined [Supplemental (D) Table 4]. The rationale of the two levels of analysis was to determine the relative proteins compositions due to the greater insolublization of proteins in CRYAAN101D lenses relative to CRYAAWT lenses (Figure 1, Table1). The level 1 determination was expected to identify the total proteins that showed insolubilization and existed in the US- and UI-protein fractions, whereas the level 2 analysis was intended to selectively identify those proteins that formed aggregates in the US- and UI-fractions. The expectation was that the information from the analyses would implicate role of specific crystallins in the aggregation and in the cataractogenic mechanism.
(i) Comparative Protein Compositions in WI-US Fractions of Lenses from CRYAAN101D and CRYYAAWT Mice
The proteins detected in the WI-US-protein fractions of CRYAAN101D lenses but were absent in the CRYYAAWT lenses were [described in Supplemental (A) Table 1]: α-enolase, ATP synthase subunit beta (mitochondrial), several histones (H1.1, H1.2, H1.3, H1.4, H2B type 1-A, H2B type 1-B, H2B type 1-C, H2B type 1-F/J/L, H2B type 1- H2B type 1-H, H2B type 1-K, H2B type 1-M, H2B type 1-P, H2B type 2B, H2B type 2E, H2B type 3A, H2B type 3B, H3.1, H3.2, H3.3, H3.3C, H4), and keratin type 1 and 2 (cytoskeleton). In contrast, the CRYYAAWT lenses contained the following proteins that were absent in the CRYAAN101D lenses (Supplemental Table 1): ezrin, gamma crystallin N, histones (2A type 1, H2A.V, H2A.Z, H2A.X), lengsin, moesin, N6- adenosine-methyltransferase subunit METTL3, pyruvate kinase PKM, radixin, retinal dehydrogenase, spectrin beta chain (non-erythrocytes), and tubulin with alpha-1A and alpha 3 chains. Together, the above results show that the WI-US fraction of CRYAAN101D lenses was enriched in several histones, which could be due to the lack of denucleation relative to CRYYAAWT lenses. Furthermore, the insolubilized proteins that could be solubilized in 8M urea from the two types of lenses were different.
(ii) Comparative Protein Compositions of WI-UI-Fractions of Lenses from CRYAAN101D- and CRYYAAWT Mice
On a comparison of the proteins present in the WI-UI-protein fractions of CRYAAN101D lenses but were absent in CRYYAAWT lenses, these were [Supplemental (B) Table 2]: ATP synthase subunit alpha and beta (mitochondrial), Cadherin-2, histones (H1.0, 1.2, 1.3, 1.4, H2A-type 1, H2A-type1-H-F, H2A-type 1-H, H2A-type 1-K, H2A type2-A, H2A type2-C, H2A type 3, H2A.J, H2AX, H2B type 1-A, H2B type 1-B, H2B type 1-C/E/G, H2B type 1-F/J/L, H2B type 1-H, H2B type 1-K, H2B type 1-M, H2B type 1-P, H2-B type 2-B, H2-B type 2-E, H2B type 3-A, H2B type 3-B, H4), keratin type II and type 6 (cytoskeletal 2 epidermal), and N6-adenosine-methyltransferase subunit METTL3. In contrast, the CRYYAAWT lenses contained the following proteins that were absent in the CRYAAN101D lenses: (Supplemental Table 2): AFG3-like protein 2, ankyrin-2, armadillo repeat protein deleted in velo-cardio-facial syndrome homolog, catenin beta-1, elongation factor 2, γD-crystallin, gap junction alpha-8 protein, glyceraldehyde-3- phosphate dehydrogenase, guanine nucleotide-binding protein [G (i) subunit alpha-1, -subunit (i) alpha-2, -G(k) subunit alpha, -G(o) subunit alpha, -G (olf) subunit alpha, -G(s) subunit alpha, -G(s) subunit alpha isoform short, -G(s) subunit alpha XLas, -G(s) subunit alpha-1, -G(s) subunit alpha-2, -G(s) subunit alpha-3, G(s) subunit alpha-12, G(s) subunit alpha-13], heat shock 70 kDa protein-1A, -1B, -1-like, heat shock cognate 71 kDa protein, histone H4, importin 5, keratin, type I cytoskeletal 13, keratin, type I cytoskeletal 19, keratin, type II cytoskeletal 71, keratin, type II cytoskeletal 72, lens epithelial cell protein LEP503, multifunctional protein ADE2, peroxiredoxin-2, phosphoglycerate kinase, pyruvate kinase PKM, Rab GDP dissociation inhibitor beta, Ras-related C3 botulinum toxin substrate 1, Ras-related C3 botulinum toxin substrate 2, Ras-related C3 botulinum toxin substrate 3, Ras-binding protein 6, retinal dehydrogenase, tubulin (alpha-1A chain, -alpha-1C chain, -beta-2A chain, - beta-2B chain, - beta-4A chain, -beta-4B chain, -beta -5A chain), and ubiquitin carboxyl-terminal hydrolase isozyme. These results again show that the majority of histones that existed in CRYAAN101D lenses were absent in the CRYYAAWT lenses, which could be due to the lack of denucleation in the former lenses. Additionally, among crystallins, specifically αB and βB2 crystallin became insoluble as their levels were higher even in the WI-UI-fraction of CRYAAN101D lenses relative to CRYYAAWT lenses.
(iii) Compositions of Aggregated Proteins (Mr >30 kDa) in WI-US- and WI-UI-Fractions of Lenses from CRYAAN101D and CRYYAAWT 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 CRYAAN101D and CRYYAAWT lenses [Supplemental (C) and (D) Tables 3 and 4]. On comparison, the proteins present as aggregates (Mr > 30 kDa) in WI-US fraction of CRYAAN101D but absent in CRYYAAWT were (Table 3): basement membrane-specific heparin sulfate proteoglycan core protein, βB3- and γC-crystallins, calcium-uptake protein 2, (mitochondrial), collagen alpha-1(IV) chain and -alpha-2(IV) chain, glial fibrillary acidic protein and nestin. In contrast, the exclusively present proteins in WI-US fraction of CRYYAAWT were: N6-adenosine-methyltransferase subunit METTL3, and γC-, γD, γE- γF-crystallins. The above list describes the selective proteins that were water insoluble-urea soluble and became part of the complexes with Mr > 30 kDa in CRYAAN101D lenses. Because the aggregated proteins contained collagen, proteoglycans, and βB3- and γC-crystallins, and calcium uptake protein 2, (mitochondrial) in the WI-US fraction of αAN101D, we hypothesize relative cellular disorganization occurred in the CRYAAN101D lenses. Similarly, the greater abundance of αA- and βB1-crystallins in the aggregated form suggested their involvement in the aggregation process along with βB3- and γC-crystallins.
On comparison of proteins that existed in WI-UI protein fraction as > 30 kDa aggregates in but in CRYAAN101D not in the CRYYAAWT were [Supplemental (D) Table 4]: γB-, γD- and γE-crystallins, N6-adenosine-methyltransferase subunit METTL3, nestin, tail-anchored protein insertion receptor WRB. In contrast, on a comparison, the proteins as >30 kDa aggregates present in the WI-UI fraction of CRYYAAWT lenses but absent in CRYAAN101D lenses were: serine/arginine repetitive matrix protein 2. In the WI-UI fraction, the greater abundance of proteins in CRYAAN101D compared to CRYYAAWT were: αA-crystallin and lens fiber major intrinsic protein. Together, the results showed that the proteins that remained urea insoluble and were associated with the membrane of CRYAAN101D lenses were: γB-, γD- and γE-crystallins, and nestin. Nestin is an intermediate filament protein, which is expressed predominantly in the developing central nervous system and skeletal muscles.
Increased Association of αAN101D with Lens Membrane in the Outer Cortical Fiber Cells relative WTαA in CRYYAAWT lenses
Our previous report (28) showed an increased levels abnormal deposition of αAN101D within the outer cortical region in CRYAAN101D lenses compared WTαA in CRYYAAWT lenses, it suggested possible relatively greater membrane binding of αAN101D. This was further investigated in experiments as described below.
(i) Immunohistochemical Analyses of Lenses from CRYAAN101D and CRYAAWT Mice
The purpose of the experiments was to determine relative levels of αAN101D and WTαA in the outer cortical regions of CRYAAN101D- vs.CRYAAWT lenses. This was examined by immunohistochemical analysis of 5-months old lenses of the two types of mice using anti-His monoclonal (for WTaA and aAN101D detection [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 CRYAAN101D mice (Shown by an arrow in Figure 4A) relative to CRYAAWT 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 CRYAAN101D lens relative to the CRYAAWT 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 CRYAAN101D lenses relative to those of CRYAAWT lenses.
(ii) Relative Membrane-Association of WTαA- and αA-N101D in Lenses of CRYAAN101D and CRYAAWT Mice
The rationale for the next experiment was that if greater membrane-association of αA-N101D occurs in vivo in CRYAAN101D lenses compared to CRYAAWT lenses, the difference in their levels could also be determined by western blot analysis in the purified membrane fractions isolated from the two types of lenses. 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 relative levels of membrane-association of αAN101D vs. WTαA in the two types of lenses could be determined by the western blot analysis. To normalize the levels of the relative association during the membrane preparations, the two lenses of 1-month-old and two lenses from 6-month old from CRYAAN101D and CRYAAWT 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. To simplify the presentation of the western blot results of fractions recovered, during the steps of membrane purification, only the results of immunoblots with anti-His antibody but not with the western blot profiles with anti-aquaporin 0 are shown in Figure 5. However, the western blot profiles with anti-aquaporin 0 of the fractions were almost identical (Results not shown). The levels of His-tagged αA (green fluorescence) in lenses of 1-month old lenses (Figure 5, left panel: WTαA [A and C] and αAN101D [B and C]) and 6-month old lenses (Figure 5, right panel: WTαA [A and C] and αAN101D [B and D]) are shown. The upper (A) and (B) profiles in both left and right panels show Coomassie blue-stained protein bands, and the lower (C) and (D) show western blot immunoreactivity with the anti-His antibody. 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 crude WS-homogenate. The results show that the green fluorescence representing WTαA in CRYAAWT 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 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 CRYAAN101D lenses. This suggests αAN101D was tightly bound and at the higher levels to lens membrane of CRYAAN101D lenses relative to CRYAAWT lenses.
On quantification of the Western blots using Image J (Figure 5, lower most panel), 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 CRYAAN101D lenses (shown in red) compared to those from CRYAAWT lenses (blue). Similarly, 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 CRYAAN101D lenses (red) compared to CRYAAWT lenses (blue). Additionally, the lane 6 (representing membrane remaining after two urea washes, right panel) of 6-month CRYAAN101D lenses exhibited about 2X greater immunoreactivity than CRYAAWT lenses (Quantification results not shown). Together, the results show that relative to CRYAAWT, higher levels of CRYAAN101D were tightly associated with the lens membranes of 1- and 6- month old CRYAAN101D mice.
(iii) Relative Membrane-Binding of Alexa 350-Labeled Recombinant WTaA- and aA-N101D Crystallins To examine whether aA-N101D 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 examined. The recombinant WT aA- and aA-N101D proteins were labeled with Alexa 350 using a protein labeling kit by the procedure described by the manufacturer (Molecular Probes, Thermofisher 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), aA-N101D 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 35-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 C57 mice) was incubated with increasing but identical concentrations of either Alexa-labelled WT aA- or aA-N101D proteins at 37οC for 6 h (See details in Methods). A relatively higher levels (>1.5X) of binding of aAN101D proteins relative to WTaA 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 αA-N101D in Lens Membranes of CRAAN101D and CRAAWT Mice
To ascertain the relative levels association αAN101D vs. WTααA 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 CRYAAN101D and CRYAAWT at 500 nm magnification and (C) and (D) from these lenses at 100 nm magnification. 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 CRYAAN101D and CRYAAWT 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 constituted about 14% and 14.2% of the total αA in the WS-and WI-proteins, respectively in the lenses of CRYAAN101d 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 CRYAAN101D lenses were about 2X more swollen relative to those of CRYAAWT lenses [Figure 7, compare (A) to (B) and (C) to (D)]. The swelling could represent water intake within the lens cells due to the potential ionic imbalance in the CRYAAN101D lenses compared to CRYAAWT 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 CRYAAN101D and CRYAAWT 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 CRYAAN101D lenses relative to CRYAAWT lenses. Next, the levels of Na, K-ATPase mRNA, and Ca2+ levels were determined in epithelial cells from lenses of CRYAAN101D and CRYAAWT mice. Both (A) and (B) in Figure 8 show intracellular Ca2+ levels in the presence of calcium orange in cultured epithelial cells from CRYAAN101D and CRYAAWT, respectively. Only a few CRYAAN101D cells showed the Ca2+ uptake, which was possibly due to our previous finding that the lens cells contained only 14% of αAN101D mutant protein . In this experiment, 100 cells from the two cultures were counted. On quantification by Image J of the number of cells that exhibited calcium orange uptake were 1.5X greater in CRYAAN101D lens cells relative to cells from CRYAAWT lenses (Figure 8B). On the determination of levels of mRNA of Na, K-ATPase in these cells, its level was 75% lower in the CRYAAN101D lens cells than CRYAAWT lens cells (Figure 9C).