Abstract
Abstract: :
Purpose: The goal of the present study is to characterize the complete structure of bovine and human MP20, the second most abundant membrane protein in the lens, to give insight into potential regulation of function and protein-protein interactions by posttranslational modifications. Methods: Membrane proteins were isolated from bovine and human lens homogenates by centrifugation following Tris and urea washes. Bovine MP20 was separated from the most abundant membrane protein, AQP0, by anion exchange chromatography performed in the presence of 1% octyl glucoside. After detergent removal and concentration, MP20 was reduced, alkylated, and cleaved with cyanogen bromide. The peptides were analyzed by HPLC-ESI-ion trap mass spectrometry and sequences confirmed by tandem mass spectrometry. Analysis of human MP20 was performed as above without purification by anion exchange chromatography. Results: Ninety-nine percent of bovine MP20 protein was observed and the C-terminal peptide, 164-173, was detected in unphosphorylated, mono-, and di-phosphorylated states. Serine 170 was phosphorylated as previously reported1 and a new site of phosphorylation was identified at threonine 171. Seventy percent of the human protein was observed from unfractionated lens membranes and the phosphorylation state of the C-terminal peptide, 159-173, existed in unphosphorylated, mono-, and di-phosphorylated forms. Quantitation of the unphosphorylated, mono-, and di-phosphorylated peptides in human lenses indicated that the relative level of phosphorylation was unchanged with age. Conclusions: The separation of MP20 from the most abundant membrane protein, AQP0, facilitated the sequence analysis of bovine MP20. This approach will permit complete characterization of structural modifications of human MP20. 1 Galvan, A., Lampe, P.D., Chung Hur, K., Howard, J.B., Eccleston, E.D., Arneson, M., Louis, C.F. (1989) J. Biol. Chem. 264,19974-19978.
Keywords: 526 protein purification and characterization • 525 protein modifications-post translational • 309 aging