Carbon turnover in the water-soluble protein of the adult human lens

Abstract

Purpose: Human eye lenses contain cells that persist from embryonic development. These unique, highly specialized fiber cells located at the core (nucleus) of the lens undergo pseudo-apoptosis to become devoid of cell nuclei and most organelles. Ostensibly lacking in protein transcriptional capabilities, it is currently believed that these nuclear fiber cells owe their extreme longevity to the perseverance of highly stable and densely packed crystallin proteins. Maintaining the structural and functional integrity of lenticular proteins is necessary to sustain cellular transparency and proper vision, yet the means by which the lens actually copes with a lifetime of oxidative stress, seemingly without any capacity for protein turnover and repair, is not completely understood. Although many years of research have been predicated upon the assumption that there is no protein turnover or renewal in nuclear fiber cells, we investigated whether or not different protein fractions possess protein of different ages by using the (14)C bomb pulse.

Methods: Adult human lenses were concentrically dissected by gently removing the cell layers in water or shaving to the nucleus with a curved micrometer-controlled blade. The cells were lysed, and the proteins were separated into water-soluble and water-insoluble fractions. The small molecules were removed using 3 kDa spin filters. The (14)C/C was measured in paired protein fractions by accelerator mass spectrometry, and an average age for the material within the sample was assigned using the (14)C bomb pulse.

Results: The water-insoluble fractions possessed (14)C/C ratios consistent with the age of the cells. In all cases, the water-soluble fractions contained carbon that was younger than the paired water-insoluble fraction.

Conclusions: As the first direct evidence of carbon turnover in protein from adult human nuclear fiber cells, this discovery supports the emerging view of the lens nucleus as a dynamic system capable of maintaining homeostasis in part due to intricate protein transport mechanisms and possibly protein repair. This finding implies that the lens plays an active role in the aversion of age-related nuclear (ARN) cataract.

Figure 1

The ¹⁴C bomb curve is recorded in biomolecules. Aboveground nuclear testing nearly doubled the level of radiocarbon (as ¹⁴CO₂) in the atmosphere between 1955 and 1963. The atmospheric ¹⁴C levels depicted by the dark blue trace are growing season averages for the northern hemisphere expressed in F¹⁴C units (fraction modern with δ¹³C fractionation correction). These ¹⁴C levels are recorded in annual plant growth and human diets. Human tissue incorporates the contemporary ¹⁴C signature of individuals’ food at the time of synthesis. Years later, specific biomolecules can be isolated and measured for ¹⁴C content to establish carbon turnover or lack thereof, as in the cellulose of tree rings.

Figure 2

Water-soluble and water-insoluble proteins from the same cells possess different ¹⁴C signatures. Fiber cells from human cadaver lenses were peeled away in concentric layers to step back in time from the periphery (youngest) to the embryonic nucleus (oldest). Each fraction containing multiple layers of cells was centrifuged to separate the soluble proteins (crystallins exclusively) from the insoluble proteins (membrane, cytoskeleton, precipitated or insoluble crystallins). Data pairs along the atmospheric record (solid black line) represent the water-soluble and water-insoluble fractions from the same cohort of cells. Vertical lines denote the year of birth for each subject born in 1933 (A) and 1962 (B). The dashed horizontal line denotes the contemporary ¹⁴C concentration in the atmosphere at the time of death. The insoluble fraction exhibits little or no carbon turnover and is correlated to the average “birth date” of the group of cells. The insoluble fractions of the inner layers of the subject born in 1933 (A) do not contain any measurable new carbon while the corresponding soluble fractions are skewed by the addition of more recent carbon in both subjects (A, B). The younger carbon in the soluble crystallins provides direct evidence of protein turnover. Error bars are smaller than the symbols, averaging ±0.005.

Figure 3

Annual carbon turnover changes the shape of the ¹⁴C bomb curve. Carbon turnover in a macromolecular sample flattens the pulse of ¹⁴C as the rate of turnover increases. When turnover reaches 0.10 (10% annually) the pulse is almost completely flattened. If turnover is less than 0.001 (0.1% annually), molecules formed before 1955 are elevated in ¹⁴C by about 2%, but turnover is difficult to detect in molecules formed after the onset of the pulse. The differences in ¹⁴C between water-soluble and water–insoluble proteins fit a model suggesting ~0.005-0.01 turnover (0.5-1% annually) of carbon in water-soluble protein.