Gupta V K, Berthoud V M, Atal N, Jarillo J A, Barrio L C, Beyer E C
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110.
Invest Ophthalmol Vis Sci. 1994 Sep;35(10):3747-58.
To identify, clone molecularly, characterize immunochemically, and express functionally a bovine lens gap junction protein (connexin).
The methods used were polymerase chain reaction, genomic cloning, RNA and DNA blotting, bacterial expression of a fusion protein, immunoblotting, alkaline phosphatase treatment, Xenopus oocyte expression, and voltage clamp technique.
A bovine genomic clone encoding a polypeptide of 44,424 d, termed connexin44 (Cx44), was isolated. Cx44 was most closely related to the lens connexins rat Cx46 and chicken Cx56. The Cx44 DNA hybridized to a 2.5 kb mRNA detected only in lens RNA. The carboxyl terminal 161 amino acids from Cx44 were expressed in bacteria fused to maltose binding protein (MBP). The Cx44/MBP fusion protein reacted in immunoblots with anti-rat Cx46(411 to 416) antibodies and with the monoclonal antibody 5H1, but not with a monoclonal antibody to MP70 nor with antibodies to other connexins. Cx44 translated in vitro from the cloned DNA showed a single band with an apparent electrophoretic mobility of approximately 50 kd on polyacrylamide gels containing sodium dodecyl sulfate. Multiple bands of 53 to 57 kd were detected by immunoblotting in homogenates of bovine lens; these bands were reduced to a broad band of approximately 50 kd by alkaline phosphatase treatment, suggesting that they represented phosphorylated forms of Cx44. Cx44 RNA injected in single oocytes induced a large and characteristic time- and voltage-dependent current. Overexpression of Cx44 produced depolarization and cell lysis. Junctional currents that could be regulated by transjunctional voltage were induced between paired oocytes injected with Cx44 RNA. Observations in paired oocytes suggested the assembly of hemichannels into junctional channels.
Cx44 is a phosphoprotein component of bovine lens fiber gap junctions. Although it has a relatively distinct sequence, it shares sequence similarity, immunologic cross-reactivity, and electrophysiological properties with rat Cx46. These data suggest that Cx44 is the protein previously identified in several immunohistochemical studies of bovine lens gap junctions that used anti-rat Cx46 antibodies. They also suggest that the formation of intercellular channels by pairing of hemichannels might prevent the cell lysis induced by the opening of unpaired hemichannels.
从分子水平鉴定、克隆、进行免疫化学特性分析并功能性表达一种牛晶状体缝隙连接蛋白（连接蛋白）。
采用的方法有聚合酶链反应、基因组克隆、RNA和DNA印迹、融合蛋白的细菌表达、免疫印迹、碱性磷酸酶处理、非洲爪蟾卵母细胞表达及电压钳技术。
分离出一个编码44424道尔顿多肽的牛基因组克隆，命名为连接蛋白44（Cx44）。Cx44与晶状体连接蛋白大鼠Cx46和鸡Cx56关系最为密切。Cx44 DNA与仅在晶状体RNA中检测到的2.5 kb mRNA杂交。来自Cx44的羧基末端161个氨基酸与麦芽糖结合蛋白（MBP）融合在细菌中表达。Cx44/MBP融合蛋白在免疫印迹中与抗大鼠Cx46（411至416）抗体及单克隆抗体5H1发生反应，但不与抗MP70单克隆抗体及其他连接蛋白抗体发生反应。从克隆DNA体外翻译的Cx44在含十二烷基硫酸钠的聚丙烯酰胺凝胶上显示出一条表观电泳迁移率约为50 kd的单带。在牛晶状体匀浆中通过免疫印迹检测到53至57 kd的多条带；经碱性磷酸酶处理后这些条带减少为一条约50 kd的宽带，表明它们代表Cx44的磷酸化形式。向单个卵母细胞注射Cx44 RNA可诱导出大的、具有特征性的时间和电压依赖性电流。Cx44的过表达导致去极化和细胞裂解。在注射了Cx44 RNA的配对卵母细胞之间诱导出可由跨连接电压调节的连接电流。在配对卵母细胞中的观察结果提示半通道组装成连接通道。
Cx44是牛晶状体纤维缝隙连接的一种磷蛋白成分。尽管它具有相对独特的序列，但与大鼠Cx46具有序列相似性、免疫交叉反应性和电生理特性。这些数据表明Cx44是先前在几项使用抗大鼠Cx46抗体的牛晶状体缝隙连接免疫组织化学研究中鉴定出的蛋白。它们还提示半通道配对形成细胞间通道可能会阻止未配对半通道开放所诱导的细胞裂解。
