Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury.
Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury.
复制标题
大鼠caspase-6的克隆、表达及其在肾缺血/再灌注损伤中的定位。
DOI：
10.1046/j.1523-1755.2002.00427.x
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Kaushal,GurP
中科院分区：
文献类型：
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作者：
Singh,AmarB;Kaushal,Varsha;Megyesi,JuditK;Shah,SudhirV;Kaushal,GurP
大鼠Caspase-6的克隆、表达及其在肾缺血/再灌注损伤中的定位研究背景Caspase-6是细胞死亡蛋白Caspase家族中的重要成员。执行者caspase是凋亡细胞中检测到的主要活性caspase，通常被认为通过裂解和失活细胞内蛋白来介导细胞凋亡的执行。然而，caspase-6mRNA和蛋白在大鼠肾脏中的表达及其在正常肾脏和疾病状态下的表达尚不清楚。方法通过筛选大鼠肾皮质λgt10cDNA文库，获得caspase-6全长基因。重组caspase-6蛋白在细菌中表达，在哺乳动物细胞中瞬时转染。结果所预测的大鼠Caspase-6的氨基酸序列为277个氨基酸，含有两个潜在的糖基化位点，即整合素结合位点(KGD)、Caspase活性位点五肽QACRG和Caspase家族标志HX2-4(S，C)X4(L，I，V，M，F)2(S，T)HG(HVDADCFVCVFLSHG)。大鼠caspase-6在已知的caspase中是独一无二的，因为它拥有一个相对较长的5‘非翻译区。在所检测的各种组织中，cas-pase-6在肾、肝、脾、心脏、肌肉、睾丸和肺中都有不同程度的表达。重组大鼠caspase-6的细菌表达导致了该酶的前体和活性形式的产生，表明该酶具有自活化作用。大鼠caspase-6在COS-1细胞中的瞬时过表达导致DNA片段化，这是细胞凋亡的标志。我们还用免疫组织化学方法检测了Caspase-6在缺血40分钟再灌注24小时肾脏中的定位和表达。正常肾脏以胞浆为主，部分肾小管可见胞核染色。缺血40min后24小时，肾脏胞浆染色较浓，弥散，胞核染色明显，提示胞浆表达增加，胞核移位。结论大鼠Caspase-6全长基因的克隆、表达、鉴定及其在正常肾脏和缺血/再灌注损伤大鼠肾脏中的定位，为进一步研究Caspase-6的表达奠定了基础。由于caspase-6参与了核基质蛋白的降解和caspase-3的激活，因此可能在肾缺血损伤中发挥重要作用。
Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury.BackgroundCaspase-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.MethodsA rat kidney cortex λgt10 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.ResultsThe 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.ConclusionsThese 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.