Expression of calpain small subunit 2 in mammalian tissues.
OBJECTIVE
The purpose of the current experiments was to more closely define the distribution and the function of calpain small subunit 2 (css2). Css2 is a newly discovered regulatory protein for the calcium activated proteases, mu- and m-calpains.


METHODS
Tissues from rat, monkey, and man of various ages were used to determine expression patterns of css2 by relative quantitative RT-PCR using 18S rRNA as an endogenous standard. Recombinant css2 and the 80 kDa catalytic subunit of m-calpain (80 kDa/css2) were co-expressed in Escherichia coli. Casein zymography was used to measure the enzymatic activity of 80 kDa/css2 proteins. Lens alpha-crystallin and beta B1-crystallin were used as substrates to determine proteolysis by 80 kDa/css2. Computer-based homology modeling was used to predict interactions between the traditional small subunit (css1) or css2 with the 80 kDa catalytic subunit.


RESULTS
Css2 appears to be a functional equivalent of css1 in vitro in that the calcium-dependent proteolytic activity of 80 kDa/css2 was similar to recombinant m-calpain (80 kDa/css1). In rat and human lens, css2 transcripts increased with age, whereas css1 transcripts decreased with age. Human beta B1-crystallin and rat alpha A-crystallin were cleaved similarly by 80 kDa/css2 and 80 kDa/css1. Interestingly, alpha A-insert crystallin was not hydrolyzed when css2 was substituted for css1 in the calpain dimer, suggesting that css2 may perform different functions from css1 in terms of proteolysis of lens crystallins during maturational growth of the lens. Css2 may also assist in the proper folding of the 80 kDa subunit and regulate protease activity in the absence of calcium.


CONCLUSIONS
The wide distribution of css2 transcripts in rat and monkey suggested that css2 is a second, widely distributed (rather than tissue-specific) calpain small subunit, in addition to the long-recognized css1. Further studies at the protein level will indicate if css2 has unique functions apart from css1.
Ma
H
H
Department of Integrative Biosciences, Oregon Health & Science University, Portland, Oregon 97239, USA. mah@ohsu.edu
Nakajima
E
E
Shih
M
M
Azuma
M
M
Shearer
T R
TR
D000293:Adolescent; D000328:Adult; D000368:Aged; D000375:Aging / Q000502:physiology; D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D003459:Crystallins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D007223:Infant; D007908:Lens, Crystalline / Q000201:enzymology*; D008253:Macaca mulatta; D008297:Male; D008875:Middle Aged; D018832:Molecular Chaperones / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014162:Transfection
D002135:Calcium-Binding Proteins; D003459:Crystallins; D018832:Molecular Chaperones; D012333:RNA, Messenger; C033668:calpastatin; D002154:Calpain