Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract: dynamic light-scattering and HPLC analysis

Abstract

Purpose: The role of oxygen in the formation of lens high-molecular-weight (HMW) protein aggregates during the development of human nuclear cataract is not well understood. The purpose of this study was to investigate lens crystallin aggregate formation in hyperbaric oxygen (HBO)-treated guinea pigs by using in vivo and in vitro

Methods: methods. Guinea pigs were treated three times weekly for 7 months with HBO, and lens crystallin aggregation was investigated in vivo with the use of dynamic light-scattering (DLS) and in vitro by HPLC analysis of water-insoluble (WI) proteins. DLS measurements were made every 0.1 mm across the 4.5- to 5.0-mm optical axis of the guinea pig lens.

Results: The average apparent diameter of proteins in the nucleus (the central region) of lenses of HBO-treated animals was nearly twice that of the control animals (P < 0.001). Size distribution analysis conducted at one selected point in the nucleus and cortex (the outer periphery of the lens) after dividing the proteins into small-diameter and large-diameter groups, showed in the O2-treated nucleus a threefold increase in intensity (P < 0.001) and a doubling in apparent size (P = 0.03) of large-diameter aggregate proteins, compared with the same control group. No significant changes in apparent protein diameter were detected in the O2-treated cortex, compared with the control. The average diameter of protein aggregates at the single selected location in the O2-treated nucleus was estimated to be 150 nm, a size capable of scattering light and similar to the size of aggregates found in human nuclear cataracts. HPLC analysis indicated that one half of the experimental nuclear WI protein fraction (that had been dissolved in guanidine) consisted of disulfide cross-linked 150- to 1000-kDa aggregates, not present in the control. HPLC-isolated aggregates contained alphaA-, beta-, gamma-, and zeta-crystallins, but not alphaB-crystallin, which is devoid of -SH groups and thus does not participate in disulfide cross-linking. All zeta-crystallin present in the nuclear WI fraction appeared to be there as a result of disulfide cross-linking.

Conclusions: The results indicate that molecular oxygen in vivo can induce the cross-linking of guinea pig lens nuclear crystallins into large disulfide-bonded aggregates capable of scattering light. A similar process may be involved in the formation of human nuclear cataract.

Figure 1

Typical slit lamp biomicroscopy photographs of guinea pig eyes. (A) A 25-month-old control animal. (B) A 25-month-old animal after 84 treatments with HBO over a 7-month period. Note the increased lens nuclear light-scattering in the O₂-treated lens, compared with the control, particularly in the very center of the nucleus (arrow). In addition, note the larger region of backscatter in the O₂-treated lens, compared with the control.

Figure 2

Typical SLS analyses in vivo of eyes of control and HBO-treated 25 month-old guinea pigs. Each analysis was conducted along the optical axis of the lens, measuring 4.5 to 5.0 mm in diameter. K counts/s: 1000 photon counts per second of light intensity. (A) Control, untreated animal; (B) animal treated 84 times with HBO. Note the increase in SLS intensity in the experimental lens, particularly in the central region, compared with the age-matched control.

Figure 3

Representative DLS analysis profiles in vivo across lenses of control and HBO-treated guinea pigs (not the same animals reported in Fig. 2). Measurements of average protein diameter were made every 0.1 mm across the approximate 5-mm optical axis of each lens. The instrument made 10 to 15 measurements of protein diameter at each point and provided an average value. The DLS data are not expressed as absolute sizes of lens proteins but as arbitrary units of diameter (see Materials and Methods for an explanation). (○) 25-month-old control animal; (♦) 25-month-old animal after 84 treatments with HBO.

Figure 4

Analysis of in vivo dynamic light-scattering data for measurements taken across the lenses of 25-month-old control and HBO-treated (84 treatments) guinea pigs. Measurements of average protein diameter were made every 0.1 mm across the approximate 5-mm optical axis of each lens (see the legend of Fig. 3 for an explanation of how this was done). These data were then averaged for three regions of one lens each for 13 control and 9 experimental animals. The total number of measurements were: anterior cortex, control: 224, experimental: 144; nucleus, control: 312, experimental: 215; and posterior cortex, control: 143, experimental: 97. Results are expressed as the mean ± SEM.

Figure 5

HPLC elution profiles of guinea pig lens WI proteins dissolved in 6 M GndHCl under nondisulfide-reducing conditions. Proteins were separated by gel filtration under non-reducing conditions at a flow rate of 240 μL/min. The profiles shown are representative of analyses of lenses from three 25-month old control animals or experimental animals treated 84 times with HBO. (A) Lens nucleus (1 mg protein applied for both the control and experimental). (B) Lens cortex (0.5 mg protein applied for both the control and experimental).

Figure 6

HPLC elution profiles for the control and experimental nuclear samples of Figure 5A (WI nuclear proteins dissolved in 6 M GndHCl) after reduction of the sample with DTT. Proteins (1 mg) were separated by gel filtration under reducing conditions at a flow rate of 240 μL/min. The profiles shown are representative of analyses of lenses from three animals 25-month-old control animals or experimental animals treated 84 times with HBO.

Figure 7

Western blot analysis of the HMW, Pk1 and Pk2 fractions contained in the experimental HPLC elution profile of Figure 5A (WI nuclear proteins dissolved in guanidine, without DTT, from the lenses of guinea pigs treated 84 times with HBO). Fractions were pooled (HMW: 22–24 minutes elution; Pk1: 25–37 minutes elution; and Pk2: 39–42 minutes elution), dialyzed, concentrated, and analyzed after reduction of disulfide with DTT. Immunostaining was conducted using antibodies to αA-, αB-, β- γ-,, and ζ-crystallins. Arrowhead and arrow: indicate polypeptides resistant to DTT reduction. Note the absence of αB-crystallin in the HMW and Pk1 fractions and the absence of ζ-crystallin in the Pk2 fraction.