Molecular Processes Implicated in Human Age-Related Nuclear Cataract

Abstract

Human age-related nuclear cataract is commonly characterized by four biochemical features that involve modifications to the structural proteins that constitute the bulk of the lens: coloration, oxidation, insolubility, and covalent cross-linking. Each of these is progressive and increases as the cataract worsens. Significant progress has been made in understanding the origin of the factors that underpin the loss of lens transparency. Of these four hallmarks of cataract, it is protein-protein cross-linking that has been the most intransigent, and it is only recently, with the advent of proteomic methodology, that mechanisms are being elucidated. A diverse range of cross-linking processes involving several amino acids have been uncovered. Although other hypotheses for the etiology of cataract have been advanced, it is likely that spontaneous decomposition of the structural proteins of the lens, which do not turn over, is responsible for the age-related changes to the properties of the lens and, ultimately, for cataract. Cataract may represent the first and best characterized of a number of human age-related diseases where spontaneous protein modification leads to ongoing deterioration and, ultimately, a loss of tissue function.

Figure 1

(A) A diagrammatic representation of the main processes underpinning ARNC formation and how this is linked to human aging. (B) At middle age, an internal barrier to diffusion forms at the anatomic zone that corresponds to the size of the lens at birth. From this age onward, GSH access to the nucleus becomes limiting. Oxygen is still able to access the nucleus, so the nuclear proteins become susceptible to oxidation. GSH levels fall. (C) Once ARNC commences, protein-protein disulfide levels increase dramatically as do levels of methionine sulfoxide, and mixed disulfides also can be detected. The lens barrier appears to be due to the large-scale binding of α-crystallin aggregates, formed by binding of α-crystallin to proteins in the lens as they denature . These attach to the inner surfaces of the fiber cell membranes, thus occluding the membrane pores. Proteins become colored, particularly in the lens center, as UV filters and other reactive metabolites (e.g., ascorbate) become bound and oxidize.

Figure 2

Racemization of Asp/Asn and Ser as a function of lens age. Over time, l-Asp and l-Asn residues in lens proteins convert to d-Asp or d-isoAsp. The degree of racemization is huge, corresponding to 1 to 2 Asp/Asn residues in every lens protein by age 60. This would be expected to lead to large-scale protein denaturation. d-Ser levels in lens proteins accumulate following a similar pattern with age, although the absolute levels are lower than those for Asp/Asn. For comparable age-matched cataract lenses, the racemization levels for both Asp/Asn and Ser are significantly higher, suggesting that spontaneous racemization plays a key role in ARNC formation. Interestingly, the levels of d-Ser are higher at age 40 to 60 years in cataract lenses, but in the older cataract lenses they do not appear to be much increased above those of age-matched normal lenses. The time zero value represents artifactual racemization due to the process of acid hydrolysis. These graphs were reproduced from data published in Hooi and Truscott.

Figure 3

Lens proteins cross-link due to the spontaneous decomposition of some amino acids: aspartic acid and asparagine. The side chains of Asp and Asn can attack the peptide bond on the C-terminal side causing peptide bond cleavage and generating a C-terminal succinimide (Asn) or a C-terminal anhydride (Asp) as shown. These cyclic intermediates can react with Lys residues to produce covalent cross-links. In addition, over time, some Asn and some Asp residues cyclize by attack of the peptide bond nitrogen atom. The internal succinimides thus formed are also susceptible to attack by Lys residues forming covalent cross-links. Since succinimides are also intermediates in isomerization and racemization, this scheme links cross-linking with isomerization/racemization.

Figure 4

Lens proteins cross-link due to the spontaneous decomposition of some amino acids: phosphoserine, cysteine, and phosphothreonine. Over time, reactive dehydroalanine (DHA) residues are formed by β-elimination of phosphoserine and cystine residues. Other protein Cys or Lys residues can then attack the DHA residues forming covalent cross-links. Phosphothreonine residues decompose in an analogous manner, forming DHB, which undergoes analogous addition reactions. DHB, dehydrobutyrine.