Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice

Yoshikuni Nakamura, Chiho Fukiage, Marjorie Shih, Hong Ma, Larry David, Mitsuyoshi Azuma, Thomas (Tom) Shearer

Research output: Contribution to journalArticle

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Abstract

PURPOSE. The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS. Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day- old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS. Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of αA-crystallin. CONCLUSIONS. Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

Original languageEnglish (US)
Pages (from-to)1460-1466
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume41
Issue number6
StatePublished - 2000

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Lenses
Proteolysis
Crystallins
Cataract
Buthionine Sulfoximine
Calcimycin
Calcium
Calcium Ionophores
Caseins
calpain Lp82
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Rodentia
Mass Spectrometry
Amino Acids
Injections
m-calpain

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice. / Nakamura, Yoshikuni; Fukiage, Chiho; Shih, Marjorie; Ma, Hong; David, Larry; Azuma, Mitsuyoshi; Shearer, Thomas (Tom).

In: Investigative Ophthalmology and Visual Science, Vol. 41, No. 6, 2000, p. 1460-1466.

Research output: Contribution to journalArticle

Nakamura, Yoshikuni ; Fukiage, Chiho ; Shih, Marjorie ; Ma, Hong ; David, Larry ; Azuma, Mitsuyoshi ; Shearer, Thomas (Tom). / Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice. In: Investigative Ophthalmology and Visual Science. 2000 ; Vol. 41, No. 6. pp. 1460-1466.
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T1 - Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice

AU - Nakamura, Yoshikuni

AU - Fukiage, Chiho

AU - Shih, Marjorie

AU - Ma, Hong

AU - David, Larry

AU - Azuma, Mitsuyoshi

AU - Shearer, Thomas (Tom)

PY - 2000

Y1 - 2000

N2 - PURPOSE. The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS. Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day- old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS. Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of αA-crystallin. CONCLUSIONS. Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

AB - PURPOSE. The purpose of the present experiments was to provide a biochemical mechanism for the involvement of lens-specific calpain Lp82 in experimental cataractogenesis in mice. METHODS. Nuclear cataracts were produced by culturing lenses from 4-week-old mice and rats in calcium ionophore A23187 or by injection of buthionine sulfoximine (BSO) into 7-day- old mice. Casein zymography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, calcium determinations, in vitro precipitation, and cleavage site analysis by mass spectrometry were performed on lens samples. RESULTS. Amino acid sequences for Lp82 were found to be highly conserved in lenses from mouse to cow, and expressed Lp82 proteolytic activity was high in the mouse and rat. Lenses from mice were more susceptible to A23187-induced cataract and BSO cataracts than rats. Both types of cataracts showed rapid elevation of calcium, activation of Lp82 and m-calpain, and proteolysis of crystallins. Lp82 caused in vitro precipitation of crystallins; and in contrast to m-calpain, Lp82 truncated only the first five amino acids from the C-terminus of αA-crystallin. CONCLUSIONS. Under pathologic conditions of massive elevation of lens calcium found in young rodent lenses, overactivation of Lp82 and m-calpain leads to rapid truncation of crystallins at both common and unique cleavage sites, precipitation of truncated crystallins, and cataract.

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