Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

Abstract

The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the waterinsoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25-and 41-yearold subjects showed distinct 5-and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52-and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25-and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. ␣A, ␣B, ␤A3, ␤A4, ␤B1, ␤B2, ␥S, and ␥D), and the second type of ␣-, ␤-, and ␥-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: ␣A-fragment (C-terminally truncated, residues 1-157), ␣B-fragment (residues 83-90), ␤B1-crystallin (residues 60 -71), ␤A3 (residues 33-44), ␤A4 (residues 106 -117), filensin (residues 78 -90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in ␣A-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.Mammalian lens contains three major structural proteins that are known as ␣-, ␤-, and ␥-crystallins. Among these, the ␣-and ␤-crystallins exist as oligomers, whereas the ␥-crystallin as a monomer. The ␣A-and ␣B-crystallins, the two subunits and primary gene products of ␣-crystallin, aggregate to form 800,000-Da oligomers, whereas ␤-and ␥-crystallins originated via gene duplication and form a superfamily. These structural proteins, by virtue of their special structural interactions and high concentrations, contribute to the transparency of the lens and provide refractive index to focus light on to the retina. With aging, crystallins show aggregation, cross-linking, and water insolubilization. Together, these processes contribute to the development of age-related lens opacity. Presently, the sequence of events that lead to development of opacity is not well understood. However, evidence suggests that a variety of posttranslational modifications in crystallins lead to their aggregation, cross-linking, and eventually water insolubilization. Because of several of the post-translational modifications simultaneously occur during aging in the native, aggregated, and water-insoluble crystallins, the identification of a single or combination of potenti...

Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation

“…Crystallin proteolysis is significantly increased in aged and cataract human lenses as compared with young human lenses (5)(6)(7)(8). Increased crystallin fragmentation in aged and cataract lenses has also been reported in nonhuman species (3, 9 -12).…”

Localization of low molecular weight crystallin peptides in the aging human lens using a MALDI mass spectrometry imaging approach

“…The extended periods that these LMW peptides remain in the lens indicate that the lens may be unable to efficiently degrade them, which can lead to unregulated accumulation of these peptides with age. These peptides emerge in the lens (~35-42 years in our study) during a period which also sees a drastic increase in WI proteins and crystallin truncation (Harrington et al, 2004;Srivastava et al, 2004), an increase in membrane associated lens proteins (Friedrich and Truscott, 2009) and the onset of a barrier to diffusion at the nucleo-cortical interface in the lens (Sweeney and Truscott, 1998). Taken together, the early middle-age is potentially an important period in lens aging, during which crystallins in the lens nucleus become extensively structurally modified and truncated.…”

“…Crystallin breakdown increases significantly in aged lenses and with the onset of cataract (Harrington et al, 2004(Harrington et al, , 2007Srivastava et al, 2004). For example, crystallin fragments (peptides) have been found in both water soluble (WS) and water insoluble (WI) fractions of the lens, with the prevalence of these fragments in the WI fraction increasing with age (Srivastava et al, 1996;Lampi et al, 1998;Harrington et al, 2007).…”

“…These age-related modifications include truncation, oxidation, glycation, deamidation and phosphorylation, amongst others (Hanson et al, 2000;Aquilina and Truscott, 2002;Harrington et al, 2004Harrington et al, , 2007Srivastava et al, 2004;Truscott, 2005;Wilmarth et al, 2006). Whilst certain structural changes appear to be associated with the normal maturation process of the lens, improper or unregulated modifications have been associated with lens opacity (Truscott et al, 1990;Truscott, 2005).…”

Quantitative DY‐maleimide‐based proteomic 2‐DE‐labeling strategies using human skin proteins

“…Comparison of 2-DE spot patterns from different samples to generate quantitative protein expression profiles allows the identification of potential biomarkers that are characteristic of a specific pathological or physiological state of a cell or tissue [4,5]. Thus, 2-DE is used to answer questions relevant to a variety of biological processes, including differentiation and development [6], aging [7,8], cancer [9], or allergy [10,11]. However, traditional, colorimetric ''protein in-gel'' visualization techniques, such as silver or CBB staining, cover only a very limited, not very sensitive dynamic range of protein expression compared with fluorescent labeling techniques.…”