亚硒酸盐诱导的白内障中大鼠晶状体主要内在蛋白的修饰

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

作者信息

Schey K L, Fowler J G, Shearer T R, David L

机构信息

Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.

出版信息

Invest Ophthalmol Vis Sci. 1999 Mar;40(3):657-67.

PMID:10067969

Abstract

PURPOSE

To identify modifications to rat lens major intrinsic protein (MIP) isolated from selenite-induced cataract and to determine whether m-calpain (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 selenite. Control and cataract lenses were harvested on day 16 and dissected into cortical and nuclear regions. Membranes were washed with urea buffer followed 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 by 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 cataractous cortical membranes, was not observed in the cataractous nuclear membranes. Mass spectrometric analysis revealed heterogeneous cleavage of the C terminus of MIP in control and cataract nuclear regions. The major site of cleavage was between residues 238 and 239, corresponding to the major site of in vitro cleavage by m-calpain. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analysis indicated that in vivo proteolysis during cataract formation also included sites closer to the C terminus not produced by m-calpain in vitro. Evidence for heterogeneous N-terminal cleavage was also observed at low levels with no differences between control and cataractous lenses. The major site of phosphorylation was determined to be at serine 235.

CONCLUSIONS

Specific sites of MIP N- and C-terminal cleavage in selenite-induced cataractous lenses were identified. The heterogeneous cleavage pattern observed suggests that m-calpain is not the sole enzyme involved in MIP C-terminal processing in rat lens nuclei.

摘要

目的

鉴定从亚硒酸盐诱导的白内障大鼠晶状体中分离出的主要内在蛋白（MIP）的修饰情况，并确定μ-钙蛋白酶（EC 3.4.22.17）是否在白内障形成过程中负责MIP的裂解。

方法

通过单次注射亚硒酸钠诱导大鼠产生白内障。在第16天收获对照和白内障晶状体，并将其解剖为皮质和核区域。用尿素缓冲液洗涤膜，然后用氢氧化钠洗涤。蛋白质进行还原/烷基化、脱脂处理，并用溴化氰（CNBr）裂解。裂解产物通过高效液相色谱（HPLC）进行分离，肽段通过质谱和串联质谱进行鉴定。通过与纯化的酶孵育进行μ-钙蛋白酶对MIP的裂解，从膜中释放的肽段通过埃德曼测序进行分析。

结果

在对照核膜和白内障皮质膜中观察到的完整C末端，在白内障核膜中未观察到。质谱分析显示对照和白内障核区域中MIP的C末端存在异质性裂解。主要裂解位点在第238和239位残基之间，对应于μ-钙蛋白酶体外裂解的主要位点。然而，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和质谱分析表明，白内障形成过程中的体内蛋白水解还包括更靠近C末端的位点，这些位点在体外不是由μ-钙蛋白酶产生的。在对照和白内障晶状体之间没有差异的低水平下也观察到了异质性N末端裂解的证据。确定主要磷酸化位点在丝氨酸235。