Posttranslational modifications in lens fiber connexins identified by off-line-HPLC MALDI-quadrupole time-of-flight mass spectrometry

PURPOSE. Gap junction intercellular communication is necessary for the development and maintenance of the lens. Lens fiber connexins are known to be posttranslationally modified, but little detail is available regarding the nature of some of these modifications and the specific amino acids affected. The purpose of this study was to identify posttranslational modification in the bovine lens fiber connexins, Cx44 and Cx49. METHODS. Crude preparations of bovine lens membranes were isolated by centrifugation. The membrane preparations were digested with trypsin or chymotrypsin, and the entire mixture of peptides produced was separated by reverse-phase high performance liquid chromatography and then analyzed by mass spectrometry and tandem mass spectrometry. RESULTS. Coverage of significant portions of the cytoplasmic domains of both Cx44 and Cx49 were successfully obtained. Several of the Ser and Thr residues in the C-tail of Cx44 were phosphorylated, whereas in Cx49 only Ser phosphorylation was detected; however, in this connexin, the phosphorylated residues were located in both the C-tail and the central cytoplasmic loop. The data also show that the N-terminal Met residue in each connexin is removed and that the newly exposed N termini become acetylated. In addition, cleavage sites were identified in both proteins. CONCLUSIONS. The study documented the nature and locations of several previously unknown posttranslational modifications in lens fiber connexins. This detailed knowledge of the specific posttranslationally modified sites will allow further work to elucidate the mechanisms that different signaling pathways use to regulate connexins in lens fiber cells.