PURPOSE - Membrane protein 20 (MP20) is the second most abundant integral membrane protein in the lens, yet little is known about its function and post-translational modifications. The purpose of this work was the determination of the primary protein structure of MP20 and the types and sites of in vivo modifications.
METHODS - Bovine MP20 was isolated by anion exchange chromatography or SDS-PAGE followed by digestion with cyanogen bromide (CNBr) or trypsin. The total membrane protein fraction was also digested with trypsin in solution. The CNBr and trypsin peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
RESULTS - Using these approaches to integral membrane protein analysis, complete sequence coverage of MP20 was obtained. The in vivo sites of phosphorylation were identified as Ser-170 and Thr-171, the latter being previously unreported. The most abundant form of MP20 is monophosphorylated at Ser-170, whereas unphosphorylated and diphosphorylated forms were in lower abundance. In addition, two sites of a rare type of glycosylation (C-mannosylation) were identified at tryptophan residues 43 and 61.
CONCLUSIONS - The functional significance of phosphorylated MP20, the predominant form, remains unknown. Glycosylation of tryptophan residues represents a new lens protein modification that can explain galectin-3 interaction and suggests a topology for MP20 in which these peptides are located in an extracellular domain.