Shih M, Lampi K J, Shearer T R, David L L
Department of Oral Molecular Biology, Oregon Health Sciences University, Portland, OR 97201, USA.
Mol Vis. 1998 Feb 27;4:4.
(1) Identify major crystallin proteins in fetal and adult bovine lens, (2) examine the N-termini of beta-crystallins for truncation, and (3) determine if the protease m-calpain (EC 3.4.22.17) is responsible for the cleavage of bovine beta-crystallins during maturation.
Crystallins from fetal and adult bovine lenses were analyzed by one and two-dimensional electrophoresis and Edman sequencing of separated proteins and their tryptic fragments. Identical techniques were used to analyze crystallins following their incubation with purified m-calpain.
The identities of the major crystallins and several additional crystallin species missing portions of their N-terminal extensions were identified in the fetal bovine lens. Besides the previously identified form of betaB1 missing 15 residues from its N-terminus, forms of betaA3 >missing 11 and 22 residues were identified. With aging, the betaA3 (-22) species became a major protein in the adult bovine lens, and minor forms of betaB2 and betaB3 missing 8 and 22 residues from their N-termini, respectively, appeared. Purified m-calpain cleaved within the N-terminal extensions of bovine beta-crystallins and removed: 12 or 15 residues from betaB1; 8 residues from betaB2; 5 or 10 residues from betaB3; and 11 or 17 residues from betaA3.
Based on the cleavage sites in vitro, m-calpain may be partially responsible for cleavage of bovine betaB1, betaB2, and betaA3 during lens maturation. However, the preference of m-calpain to remove 12 residues from betaB1, and 11 and 17 residues from betaA3, suggested that the betaB1 (-15) and betaA3 (-22) species found in vivo were produced by a different protease. This unidentified protease may have a preference for the asparagine-proline-X-proline sequence found in the N-terminal extensions of betaB1 and betaA3.
（1）鉴定胎牛和成年牛晶状体中的主要晶状体蛋白；（2）检测β-晶状体蛋白的N端是否存在截短；（3）确定蛋白酶m-钙蛋白酶（EC 3.4.22.17）是否在成熟过程中负责牛β-晶状体蛋白的裂解。
通过一维及二维电泳、对分离出的蛋白质及其胰蛋白酶片段进行埃德曼测序，分析胎牛和成年牛晶状体中的晶状体蛋白。采用相同技术分析纯化的m-钙蛋白酶孵育后的晶状体蛋白。
在胎牛晶状体中鉴定出主要晶状体蛋白以及几种N端延伸部分缺失的额外晶状体蛋白种类。除了之前鉴定出的N端缺失15个残基的βB1形式外，还鉴定出缺失11个和22个残基的βA3形式。随着年龄增长，βA3（-22）种类成为成年牛晶状体中的主要蛋白质，并且出现了分别在N端缺失8个和22个残基的βB2和βB3的次要形式。纯化的m-钙蛋白酶在牛β-晶状体蛋白的N端延伸区内裂解并去除：βB1的12个或15个残基；βB2的8个残基；βB3的5个或10个残基；以及βA3的11个或17个残基。
基于体外裂解位点，m-钙蛋白酶可能在晶状体成熟过程中部分负责牛βB1、βB2和βA3的裂解。然而，m-钙蛋白酶倾向于从βB1中去除12个残基，从βA3中去除11个和17个残基，这表明体内发现的βB1（-15）和βA3（-22）种类是由不同的蛋白酶产生的。这种未鉴定的蛋白酶可能对βB1和βA3的N端延伸区中发现的天冬酰胺-脯氨酸-X-脯氨酸序列有偏好。
