Contribution of calpain Lp82-induced proteolysis to experimental cataractogenesis in mice.
OBJECTIVE
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 alphaA-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.
Nakamura
Y
Y
Research Laboratories, Senju Pharmaceutical, Kobe, Japan.
Fukiage
C
C
Shih
M
M
Ma
H
H
David
L L
LL
Azuma
M
M
Shearer
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D019328:Buthionine Sulfoximine; D000001:Calcimycin; D002118:Calcium / Q000378:metabolism; D002154:Calpain; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D008027:Light; D013058:Mass Spectrometry; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D012542:Scattering, Radiation; D013054:Spectrophotometry, Atomic
D000001:Calcimycin; D019328:Buthionine Sulfoximine; D002154:Calpain; D003546:Cysteine Endopeptidases; C118624:calpain Lp82; D002118:Calcium