Characterization and regulation of lens-specific calpain Lp82.
Eye tissues contain splice variants of muscle-preferred p94 (calpain 3), such as lens-specific Lp82 and Lp85, retina-specific Rt88, and cornea-specific Cn94. The purpose of the present experiment was to analyze the activation and regulation of the best characterized p94 splice variant, Lp82. Recombinant rat Lp82 (rLp82) was expressed using the baculovirus system, purified with Ni-NTA affinity and DEAE-ion exchange chromatographies, and characterized by SDS-PAGE, casein zymography, and immunoblotting. After incubation with calcium, rLp82 autolyzed into two major fragments at approximately 60 and 22 kDa. Sequencing of the autolytic fragments showed loss of three amino acids from the N terminus and cleavage near the IS2 region. Also, Lp82 and calpain 2 were found to hydrolyze each other. Calpastatin inhibited calpain 2 activity, but not Lp82. Homology modeling suggested that the lack of inhibition of Lp82 by calpastatin was due to molecular clashes at the unique AX1 region of Lp82. Lp82 also hydrolyzed calpastatin. These results suggested that Lp82 might regulate other calpain activities and cause hydrolysis of substrates such as crystallins during lens cataract formation.
Fukiage
Chiho
C
Department of Oral Molecular Biology and Ophthalmology, Oregon Health and Science University, Portland, Oregon 97201, USA. fukiagec@ohsu.edu
Nakajima
Emi
E
Ma
Hong
H
Azuma
Mitsuyoshi
M
Shearer
Thomas R
TR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D007908:Lens, Crystalline / Q000201:enzymology*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D002154:Calpain; C118624:calpain Lp82