Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury.
BACKGROUND
Caspase-6 is an important member of the executioner caspases in the caspase family of cell death proteases. The executioner caspases are the major active caspases detected in apoptotic cells and are generally considered to mediate the execution of apoptosis by cleaving and inactivating intracellular proteins. However, the complete characterization of mRNA and protein of caspase-6 in rat and its expression in normal kidney and in disease state has not been previously elucidated.


METHODS
A rat kidney cortex lambdagt10 cDNA library was screened to isolate the full-length caspase-6 cDNA. The recombinant caspase-6 protein was characterized by expression in bacteria and by transient transfection in mammalian cells. The expression in various tissues was analyzed by Northern blot, and localization in normal and ischemic kidney was performed by immunohistochemistry.


RESULTS
The predicted amino acid sequence of rat caspase-6 contains 277 amino acids, with two potential glycosylation sites, an integrin binding site (KGD), the caspase active site pentapeptide QACRG and the caspase family signature, HX2-4(S,C) X4(L,I,V,M,F)2(S,T)HG (HVDADCFVCVFLSHG). Rat caspase-6 is unique among known caspases by possessing a relatively long 5' untranslational region. Among various tissues tested, cas-pase-6 was expressed in varying levels in kidney, liver, spleen, heart, muscle, testis, and lung. Bacterial expression of recombinant rat caspase-6 resulted in production of both of the pro-form and active form of the enzyme suggesting autoactivation. Transient overexpression of rat caspase-6 in COS-1 cells induced DNA fragmentation, a hallmark of apoptosis. We also examined the localization and expression of caspase-6 by immunohistochemistry in kidneys subjected to 40 minutes of ischemia followed by 24 hours of reperfusion injury. Normal kidney showed mostly cytoplasmic and some nuclear staining of the tubules. Kidneys 24 hours after 40 minutes of ischemia showed more intense and diffused cytoplasmic staining with prominent nuclear staining, indicating increased expression and translocation from the cytoplasm to the nuclei. The staining in glomeruli was negative in both normal and ischemic kidney.


CONCLUSIONS
These studies demonstrate cloning, expression and characterization of the full-length rat caspase-6 and its localization in normal kidneys and kidneys subjected to ischemia/reperfusion injury. Since caspase-6 is involved in the degradation of nuclear matrix proteins and in activation of caspase-3, it may play an important role during renal ischemic injury.
Singh
Amar B
AB
Department of Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205, USA.
Kaushal
Varsha
V
Megyesi
Judit K
JK
Shah
Sudhir V
SV
Kaushal
Gur P
GP
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015152:Blotting, Northern; D053178:Caspase 6; D020169:Caspases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D007150:Immunohistochemistry; D007511:Ischemia / Q000201:enzymology*; D007668:Kidney / Q000098:blood supply* / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology*; D014018:Tissue Distribution
C505518:Casp6 protein, rat; D053178:Caspase 6; D020169:Caspases