The Major in vivo Modifications of the Human Water-insoluble Lens Crystallins are Disulfide Bonds, Deamidation, Methionine Oxidation and Backbone Cleavage

Kirk

Journal of Biological Chemistry

2004

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...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Covalent Multimers of Crystallins in Aging Human Lenses

Kirk

Journal of Biological Chemistry

2004

“…3). Cataracts are a common problem in the elderly, with progressive deposition of insoluble protein in the lens and extensive oxidative damage generally caused by environmental factors such as UV exposure [104][105].…”

Section: The Ocular Lens and Vitreous Humor In Alzheimer's Diseasementioning

confidence: 99%

Ocular Biomarkers for Early Detection of Alzheimer's Disease

Frost

Martins

Kanagasingam

2010

JAD

Alzheimer's disease (AD) is the most common form of dementia and is characterized clinically by a progressive decline in memory, learning and executive function and neuropathologically by the presence of cerebral amyloid deposits. Despite a century of research, there is still no cure or conclusive pre-mortem diagnosis for the disease. A number of symptom-modifying drugs for AD have been developed, but their efficacy is minimal and short-lived. AD cognitive symptoms arise only after significant, irreversible neural deterioration has occurred, hence there is an urgent need to detect AD early, before the onset of cognitive symptoms. An accurate, early diagnostic test for AD would enable current and future treatments to be more effective, as well as contribute to the development of new treatments.While most AD related pathology occurs in the brain, the disease has also been reported to affect the eye, which is more accessible for imaging than the brain. AD-related proteins exist in the normal human eye and may produce ocular pathology in AD. There is some homology between the retinal and cerebral vasculatures and the retina also contains nerve cells and fibers that form a sensory extension of the brain. The eye is the only place in the body where vasculature or neural tissue is available for non-invasive optical imaging. This article presents a review of current literature on ocular morphology in AD and discusses the potential for an ocular based screening test for AD.

“…Cataractogenesis is thought to be a result of accumulation of insoluble aggregates and cross-linked products of the different forms of crystallins (40). Deamidation of crystallin is one of the most common posttranslational modifications that occur during cataract formation (41,42). Recently, it was demonstrated that crystallins in which asparagines are replaced with aspartates to mimic deamidation aggregate to a much larger extent than do wild-type crystallins and display other characteristics that are associated with cataract formation.…”

Section: Asparagine Deamidation In Disease-dysregulation Of a Normal mentioning

confidence: 99%

Chronoregulation by Asparagine Deamidation

Weintraub

Deverman

2007

Sci. STKE

Every asparagine in every protein undergoes nonenzymatic deamidation to aspartate or isoaspartate at a rate determined by the surrounding protein structure and cellular environment. Under physiologic conditions, the deamidation half-life of individual asparagines in proteins is proposed to range from less than a day to several centuries. More than 200 proteins have been shown to undergo deamidation to a meaningful degree, and modeling predicts that hundreds more undergo deamidation at rates that have the potential to be of biological consequence. Because deamidation converts an asparagine into an aspartate or isoaspartate, it introduces a negative charge into a protein and results in the isomerization of a residue. Therefore, deamidation has the potential to change protein function. Additionally, deamidation is thought to render some proteins more susceptible to degradation. In most instances in which asparagine deamidation has been identified in vivo, it is involved in pathology. Hence, deamidation has been viewed primarily as a form of protein damage. However, the pervasiveness and evolutionary persistence of these unstable asparagines suggest that they may have a beneficial role. Notably, the change of even a single neighboring amino acid can have a marked effect on the rate of deamidation of an asparagine. Therefore, the underlying rate of deamidation of any asparagine is genetically programmable. This characteristic, combined with the wide range of deamidation rates that can be programmed, imparts to asparagines the potential to serve as molecular timers that regulate protein function and stability.