Identification and characterization of a retina-specific calpain (Rt88) from rat.
OBJECTIVE
To identify and characterize a newly discovered calpain termed Rt88 from rat retina.


METHODS
Rt88 in retina under normal physiological conditions was characterized in Sprague-Dawley rats of various ages by competitive RT-PCR, Northern blot analysis, cDNA cloning and sequencing. Recombinant Rt88 was expressed in the baculovirus system and characterized by casein zymography and immunoblotting.


RESULTS
Rt88 was sequenced and found to be similar to muscle calpain p94 except for three differences. A different exon 1 (as in lens Lp82 calpain) was present, and exons 15 and 16 in the unique IS2 region of muscle p94 were deleted. Of eleven tissues studied, mRNA for Rt88 was found only in retina where Rt88 increased with maturation and then remained constant. Casein zymography showed that rRt88 was proteolytically active after activation by calcium, but intact rRt88 was rapidly broken due to the presence of the IS1 region in domain II.


CONCLUSIONS
Rt88 is a retina-specific, calcium activated protease from the calpain superfamily (EC 3.4.22.17) of cysteine proteases. Rt88 is a recently identified member of the AX1 subfamily of calpains showing alternative exon 1 usage. So far, all AX1 subfamily members are from eye. Rt88 may perform specific proteolytic functions during development, normal turnover, or pathological degeneration of retinal proteins.
Azuma
M
M
Department of Oral Molecular Biology and Ophthalmology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Fukiage
C
C
Higashine
M
M
Nakajima
T
T
Ma
H
H
Shearer
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002154:Calpain / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D005136:Eye Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D005136:Eye Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D002154:Calpain; C078134:calpain p94