Tracking pathology with proteomics: Identification ofin vivo degradation products of αB-crystallin

Colvis, Christine M.; Duglas-Tabor, Yvonne; Werth, Karena B.; Vieira, Nancy E.; Kowalak, Jeffrey A.; Janjani, Alireza; Yergey, Alfred L.; Garland, Donita

doi:10.1002/1522-2683(20000601)21:11<2219::aid-elps2219>3.0.co;2-r

Cited by 17 publications

(6 citation statements)

References 11 publications

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“…Elevated amounts of truncated αB 1-174 have been observed in cataracts from some human patients 11, in Soemmerring’s ring “after” cataract 36, and in an infant with persistent hyperplastic primary vitreous where cells invade the posterior lens 11. A major finding of the present investigation was that elevation of lenticular calcium by A23187 also produced αB 1-174 in cultured human lenses, and that the process could be reproduced in cultured monkey lenses.…”

Section: Discussionsupporting

confidence: 61%

Human and Monkey Lenses Cultured with Calcium Ionophore Form αB-Crystallin Lacking the C-Terminal Lysine, a Prominent Feature of Some Human Cataracts

Nakajima

David

Riviere³

et al. 2009

Invest. Ophthalmol. Vis. Sci.

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Purpose Elevation of lens calcium occurs in both human and experimental animal cataracts, and opacification may result from calcium-activated proteolysis. The purpose of the present study was to determine if calcium accumulation in cultured human and Macaca mulatta lenses results in proteolysis of crystallins, the major lens proteins. Methods Two-dimensional electrophoresis and mass spectrometry were used to construct detailed maps of human and monkey lens crystallins so that proteolysis following calcium accumulation could be monitored and the altered crystallins identified. Human and macaque lenses cultured in A23187 showed elevated lenticular calcium and superficial cortical opacities. The carboxypeptidase E (CPE) gene is expressed in human lens, and its presence in lens fibers was demonstrated by western blot. To investigate if CPE could cause similar truncation, purified αB-crystallin and CPE were incubated in vitro Results The major change observed in the crystallins of these cultured lenses was the accumulation of αB1-174-crystallin resulting from the loss of a C-terminal lysine. This result was significant, because similar appearance of αB1-174 is a prominent change in some human cataracts. αB-crystallin and CPE incubation result in the formation αB1-174-crystallin. This truncation was specific to αB1-174-crystallin since other crystallins were not proteolyzed. While a weaker activator than zinc, calcium was capable of activating CPE in vitro. Conclusions Since zinc concentrations did not increase during culture in A23187, calcium uptake in lens may be responsible for CPE activation and αB1-174 formation during cataract.

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Section: Discussionsupporting

confidence: 61%

Human and Monkey Lenses Cultured with Calcium Ionophore Form αB-Crystallin Lacking the C-Terminal Lysine, a Prominent Feature of Some Human Cataracts

Nakajima

David

Riviere³

et al. 2009

Invest. Ophthalmol. Vis. Sci.

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“…The first dimension isoelectric focusing was performed using a BioRad Protean IEF Cell with 7 cm 3-10NL immobilized pH gradient strips (Colvis et al, 2000). Frozen lens samples were homogenized on ice with 8 M urea containing 4% CHAPS and 100 mM DTT (rehydration buffer).…”

Section: D Gel Electrophoresismentioning

confidence: 99%

Pb2+ exposure alters the lens αA-crystallin protein profile in vivo and induces cataract formation in lens organ culture

Neal¹,

Aykin-Burns²,

Erçal³

et al. 2005

Toxicology

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“…3c). ␣B 1-170 and ␣B 1-163 were also found in human lens fiber cells growing in lens capsules following cataract surgery (21). This suggested that calpains are also active in human lens.…”

Section: Table I Masses and Identities Of Truncated ␣-Crystallins Promentioning

confidence: 99%

Mass Measurements of C-terminally Truncated α-Crystallins from Two-dimensional Gels Identify Lp82 as a Major Endopeptidase in Rat Lens

Ueda

Fukiage

Shih

et al. 2002

Molecular & Cellular Proteomics

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Molecular chaperone activity of lens ␣-crystallins is reduced by loss of the C terminus. The purpose of this experiment was to 1) determine the cleavage sites produced in vitro by ubiquitous m-calpain and lens-specific Lp82 on ␣-crystallins, 2) identify ␣-crystallin cleavage sites produced in vivo during maturation and cataract formation in rat lens, and 3) estimate the relative activities of Lp82 and m-calpain by appearance of protease-specific cleavage products in vivo. Total soluble protein from young rat lens was incubated with recombinant m-calpain or Lp82 and 2 mM Ca 2؉ . Resulting fragmented ␣-crystallins were separated by two-dimensional gel electrophoresis. Eluted ␣-crystallin spots were analyzed by mass spectrometry. Cleavage sites on insoluble ␣-crystallins were determined similarly in mature rat lens nucleus and in cataractous rat lens nucleus induced by selenite. In vitro proteolysis of ␣A-crystallin by Lp82 and m-calpain produced unique cleavage sites by removing 5 and 11 residues, respectively, from the C terminus. In vivo, the protease-specific truncations removing 5 and 11 residues from ␣A were both found in maturing lens, whereas only the truncation removing 5 residues was found in cataractous lens. Other truncation sites, common to both calpain isoforms, resulted from the removal of 8, 10, 16, 17, and 22 residues from the C terminus of ␣A. Using uniquely truncated ␣A-crystallins as in vivo markers, Lp82 and m-calpain were both found to be active during normal maturation of rat lens, whereas Lp82 seemed especially active during selenite cataract formation. These C-terminal truncations decrease chaperone activity of ␣-crystallins, possibly leading to the observed increases in insoluble proteins during aging and cataract. The methodology that allowed accurate mass measurements of proteins eluted from 2D gels should be useful to examine rapidly other post-translational modifications. ␣-Crystallins in lens are related to the small heat shock family of proteins (1). Because lens proteins have very little turnover, ␣-crystallin functions as a molecular chaperone to prevent aggregation of other lens proteins (2). The C terminus is essential, because loss of 16 amino acid residues of C terminus from ␣A-crystallin caused a loss of 50% of its chaperone activity (3).Truncation of lens crystallins is a common feature of both aging and cataract formation in rodents. A number of experiments revealed that calcium-activated proteases (calpains) were involved in formation of cataracts induced by selenite (4), galactose (5), diamide (6), and the hypocholesterolemic drug U18666A (7) and in hereditary rat Shumiya cataract (8). One of the ubiquitous calpains, m-calpain, has been credited with the proteolysis of ␣-and ␤-crystallins during cataractogenesis in rodents. Incubation of ␣A-crystallin with m-calpain reduced chaperone activity and produced truncated forms of ␣A that migrated during two-dimensional electrophoresis (2-DE) 1 to positions similar to truncated forms of ␣A observed in cataractous lenses (9). A re...

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Tracking pathology with proteomics: Identification ofin vivo degradation products of αB-crystallin

Cited by 17 publications

References 11 publications

Human and Monkey Lenses Cultured with Calcium Ionophore Form αB-Crystallin Lacking the C-Terminal Lysine, a Prominent Feature of Some Human Cataracts

Human and Monkey Lenses Cultured with Calcium Ionophore Form αB-Crystallin Lacking the C-Terminal Lysine, a Prominent Feature of Some Human Cataracts

Pb2+ exposure alters the lens αA-crystallin protein profile in vivo and induces cataract formation in lens organ culture

Mass Measurements of C-terminally Truncated α-Crystallins from Two-dimensional Gels Identify Lp82 as a Major Endopeptidase in Rat Lens

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