Large-scale binding of α-crystallin to cell membranes of aged normal human lenses: a phenomenon that can be induced by mild thermal stress

Abstract

Purpose: With age, large amounts of crystallins become associated with fiber cell membranes in the human lens nucleus, and it has been proposed that this binding of protein may lead to the obstruction of membrane pores and the onset of a barrier to diffusion. This study focused on membrane binding within the barrier region and the outermost lens cortex.

Methods: Human lenses across the age range were used, and the interaction of crystallins with membranes was examined using sucrose density gradient centrifugation, two-dimensional gel electrophoresis, and amine-reactive isobaric tagging technology. Lipids were quantified using shotgun lipidemics.

Results: Binding of proteins to cell membranes in the barrier region was found to be different from that in the lens nucleus because in the barrier and outer cortical regions, only one high-density band formed. Most of the membrane-associated protein in this high-density band was α-crystallin. Mild thermal stress of intact young lenses led to pronounced membrane binding of proteins and yielded a sucrose density pattern in all lens regions that appeared to be identical with that from older lenses.

Conclusions: α-Crystallin is the major protein that binds to cell membranes in the barrier region of lenses after middle age. Exposure of young human lenses to mild thermal stress results in large-scale binding of α-crystallin to cell membranes. The density gradient profiles of such heated lenses appear to be indistinguishable from those of older normal lenses. The data support the hypothesis that temperature may be a factor responsible for age-related changes to the human lens.

Figure 1.

Regional and age differences in the human lens as determined by sucrose density centrifugation. (a) 34-year-old lens and (b) 74-year-old lens dissected into outer, barrier, inner, and core regions. (c) Diagram illustrating the four regions of the lens obtained by dissection.

Figure 2.

Sucrose density gradient patterns as a function of age in (a) the barrier and (b) the outer regions of the lens. Lens ages are shown at the bottom of each tube.

Figure 3.

Densitometric analysis of protein at each interface of the sucrose gradients as a function of age. (a) SG6, (b) SG6a, (c) SG5, (d) SG4, (e) SG3, and (f) SG2. Barrier (●) and outer (○) regions.

Figure 4.

Amount of sphingomyelin detected at (a) SG5, (b) SG4, (c) SG3, and (d) SG2 as a function of age in the barrier region.

Figure 5.

Two dimensional SDS-PAGE gels of SG2 isolated from the barrier regions of a (a) 53-year-old and a (b) 73-year-old lens. The identities of spots were confirmed using in-gel tryptic digestion followed by mass spectrometry.

Figure 6.

Changes in the relative abundance of lens crystallins in (a) SG2 from the barrier region and (b) SG1 and SG2 from the core. The four-plex iTraq technology technique was used to compare the relative amount of each crystallin present in SG2 (or SG1 in b) from a 71- and a 72-year-old lens with the amount present in the same fraction isolated from a 49- and a 50-year-old lens. Error bars represent the combined SD between age groups from duplicate experiments. In this method of analysis, a value of 1 on the y-axis means that there has been no change in relative abundance of that protein between young and old groups. A value >1 indicates a larger relative amount of that polypeptide.

Figure 7.

The effect of mild thermal stress on the appearance of sucrose density gradient profiles. Sucrose gradients of the four regions of a (a) 31-year-old lens pair and a (c) 75-year-old lens pair before (left) and after (right) incubation at 50°C for 15 hours. (b, d) Sphingomyelin content at each interface of the sucrose gradients from the core regions before and after incubation.