University of Wollongong. School of Biological Sciences


The crystalline lens, with its unique protein content and cellular architecture, providesthe refractive index required to focus light on the retina. Mammalian lens fibre cellscontain high concentrations of structural proteins known as α-, β- and γ-crystallins,which account for approximately 90% of total protein mass in the lens. α-Crystallinalso functions as a molecular chaperone by maintaining structurally compromised lens proteins in stable, soluble complexes. With age, crystallins undergo a multitude of post translational modifications, which contribute to their destabilization. As a result, the pool of soluble, uncomplexed α-crystallin in the lens is exhausted by age 40. In the absence of a functional chaperone system, aggregation and association with the cellular membrane/cytoskeleton increases significantly with age. These changes to lenticular crystallins are believed to be the major contributors to lens opacification. This thesis examines age-related modifications of crystallins and cytoskeletal proteins in the lens. A large number of endogenous low molecular weight (LMW) peptides, derived from the breakdown of crystallins, were characterized in the human lens using mass spectrometry. LMW peptides originating from α-crystallinwere found to cover almost the entire amino acid sequence of the full length protein. Peptides which originated from β-crystallin subunits and γS-crystallin were alsoidentified, but not from other γ-crystallin subunits. The large diversity of these LMW peptides indicates that crystallins undergo extensive cleavage with age, and that thelens is incapable of removing or completely degrading these products. Analysis of the terminal residues of these LMW peptides suggested that the breakdown of lenticular crystallins may arise from both trypsin-like proteolysis and non-enzymatic cleavages,with subsequent cleavages accounting for the sequential loss of terminal residues of these peptides. Utilizing the powerful methodology of matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), several LMW peptides were spatially mapped in situ across thin cryosections of human lenses. It was observed that four peptides, derived from αA-, αB- and γS-crystallins, appeared predominantly in the nuclear fibre cells, and were only found in the cortical fibrecells of older lenses. In contrast, another major peptide, derived from the C-terminalbreakdown of βA3-crystallin, was present in the cortical and nuclear regions of both young and old lenses.

This thesis also reports on a mixture of cross-linked water-soluble complexes in the lens. When digested, these protein complexes of 40-50 kDa were found to consist of fragments from multiple crystallin subunits, with βB1, βA3 and γS the dominant species. Remarkably, these crystallin fragments were similar to the LMW crystallin content present in the urea-soluble lens fraction. This suggests that there may be two separate pathways operating in the aging lens; crystallin fragments either become insoluble through interactions with cytoskeletal/membrane components, or formcross-linked water-soluble complexes.

In a series of binding assays using fluorescently-labelled α-crystallin subunits, the binding capacity of α-crystallin for both bovine and human lens membranes was determined. It was found that the human lens nuclear membrane had the highest binding capacity for both αA- and αB-crystallins, suggesting that the structural components of the lens nuclear membrane may be significantly different from that ofthe lens cortical membranes. Also, intact α-crystallin remain bound to the lens membrane even after the membranes were vigorously stripped with alkaline solution, demonstrating that α-crystallin may be an intrinsic component of the fibre cellmembrane. Moreover, in a series of pull-out studies using a recombinant maltose-binding protein-αB-crystallin (MBP-αB) fusion protein, α-crystallin was found to associate with both the intact and breakdown products of lens cytoskeletal proteins. The N-terminal region of filensin, in particular, appeared to be a major region of this protein which α-crystallin associates with. These results demonstrate that the interaction between α-crystallin and the lens membrane/cytoskeleton is potentially a mechanism by which lenticular crystallins become insoluble with age. Together, the findings of this thesis provide important insights into the majorage-related changes of proteins in the human lens.