Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA.
Exp Eye Res. 2009 Dec;89(6):898-904. doi: 10.1016/j.exer.2009.07.015. Epub 2009 Jul 29.
Connexins 46 and 50 combine to form the gap junctions in ocular lens fiber cells. These proteins are known to be modified with fiber cell age; however, limited work has been done to characterize specific lens connexin modifications. In this report, bovine lens membrane proteins were isolated, digested by multiple enzymes, and analyzed by HPLC-tandem mass spectrometry. Automated database searching revealed the locations of both phosphorylation and truncation sites. The results confirmed the full sequence of connexin 46 and 99% of the connexin 50 sequence. Eighteen phosphorylation sites on connexin 50 and nine phosphorylation sites on connexin 46 were identified, all on serine or threonine residues. All but three phosphorylation sites on connexin 50 were located the cytoplasmic C-terminus. All of the truncation sites of connexin 50 were localized in the cytoplasmic C-terminus (region 280-304). Truncation sites in connexin 46 were found in four different regions including: the N-terminus (residue G2), the cytoplasmic loop (residues 121-124), the cytoplasmic C-terminus (residues 251-285), and the distal C-terminus (residues 344-395). In an analysis of dissected lenses some truncation sites were specific to nucleus samples and others were detected in both nucleus and cortex samples.
连接蛋白 46 和 50 结合形成了眼晶状体纤维细胞中的间隙连接。已知这些蛋白随纤维细胞年龄而改变，但对特定的晶状体连接蛋白改变的特征研究有限。在本报告中，我们分离了牛晶状体膜蛋白，用多种酶消化，并用 HPLC-串联质谱法进行分析。自动数据库搜索揭示了磷酸化和截断位点的位置。结果证实了连接蛋白 46 的完整序列和连接蛋白 50 的 99%序列。在连接蛋白 50 上鉴定出 18 个磷酸化位点和连接蛋白 46 上的 9 个磷酸化位点，均位于丝氨酸或苏氨酸残基上。连接蛋白 50 上除三个磷酸化位点外的所有位点均位于细胞质 C 末端。连接蛋白 50 的所有截断位点均位于细胞质 C 末端（280-304 区）。连接蛋白 46 的截断位点存在于四个不同的区域，包括：N 端（残基 G2）、细胞质环（残基 121-124）、细胞质 C 端（残基 251-285）和远端 C 端（残基 344-395）。在对分离晶状体的分析中，一些截断位点仅存在于核样本中，而另一些则存在于核和皮质样本中。
