Modifications to rat lens major intrinsic protein in selenite-induced cataract

PURPOSE. To identify modifications to rat lens major intrinsic protein (MIP ) isolated from selenite-induced cataract and to determine whether m-calpai n (EC 3.4.22.17) is responsible for cleavage of MIP during cataractogenesis . METHODS. Cataracts were induced in rats by a single injection of sodium sel enite. Control and cataract lenses were harvested on day 16 and dissected i nto cortical and nuclear regions. Membranes were washed with urea buffer fo llowed by NaOH. The protein was reduced/alkylated, delipidated, and cleaved with cyanogen bromide (CNBr). Cleavage products were fractionated by high- performance liquid chromatography (HPLC), and peptides were characterized b y mass spectrometry and tandem mass spectrometry. MIP cleavage by m-calpain was carried out by incubation with purified enzyme, and peptides released from the membrane were analyzed by Edman sequencing. RESULTS. The intact C terminus, observed in the control nuclear and catarac tous cortical membranes, was not observed in the cataractous nuclear membra nes. Mass spectrometric analysis revealed heterogeneous cleavage of the C t erminus of MIP in control and cataract nuclear regions. The major site of c leavage was between residues 238 and 239, corresponding to the major site o f in vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacryl amide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to t he C terminus not produced by nl-calpain in vitro. Evidence for heterogeneo us N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation w as determined to be at serine 235. CONCLUSIONS. Specific sites of MIP N- and C-terminal cleavage in selenite-i nduced cataractous lenses were identified. The heterogeneous cleavage patte rn observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.