SEA组件174-201位氨基酸对大鼠黏蛋白rMuc3羧基端酶切的影响

Influence of 174-201 residues of SEA module on the proteolytic cleavage of rodent Muc3 C-terminal domain

目的探讨大鼠黏蛋白3(rMuc3)羧基端SEA组件内174-201位氨基酸对LSKGSIVV基序酶切的影响。方法采用ClustalX1.83软件对包含SEA组件的蛋白质进行比对,选择SEA组件后续79个氨基酸中保守的氨基酸残基,利用定点突变技术将这些保守残基突变为终止密码子,分别命名为p20t1、p20t2、p20t3、p20t4,并测序验证。将不同长度的突变体转染COS-7细胞,用N-端V5标签抗体行Western blotting,检测各突变体的酶切情况。结果rMuc3羧基端第174位丝氨酸(S)、第201位半胱氨酸(C)、第212位酪氨酸(Y)、第223位酪氨酸(Y)在多种含SEA组件的蛋白质中非常保守。Western blotting检测结果表明,含rMuc3羧基端的p20及突变体p20t2、p20t3、p20t4的表达产物在LSKGSIVV基序处均能发生酶切,均可检测到30kD的酶切后片段,但p20t1的表达产物在LSKGSIVV位点处不能发生蛋白酶切。结论rMuc3羧基端第174位S至第201位C残基对LSKGSIVV基序酶切的发生至关重要,是SEA组件在LSKGSIVV基序发生蛋白酶切的另一必要条件。

Objective To explore the influence of 174-201 residues in the SEA module on the proteolytic cleavage of LSKGSIVV amino acid sequence in rodent Muc3 （rMuc3） C-terminal domain. Methods Several SEA module-containing proteins were compared with Clustalx 1.83 software to get the conserved amino acid residues. Among the ultimate seventy-nine amino acids of SEA module,the conserved amino acids were mutated to stop codon （TAA）,and they were named p20t1,p20t2,p20t3 and p20t4,respectively,by site-directed mutation. The mutation results were confirmed by sequencing. These mutants with different length were transfected into COS-7 cells,and were detected by Western blotting using anti-V5 antibody which could recognize the V5 epitope residing at N-terminal. Results Serine （174th situs）,cysteine （201st situs）,tyrosine （212th situs） and tyrosine （223rd situs） among the different SEA module-containing proteins were most conserved. The wild type of rMuc3 SEA carboxyl terminal domain from p20 and the products from p20t2,p20t3 and p20t4 were all cleaved at LSKGSIVV motif as described. The 30kD fragments were detected in all the mutants mentioned above after the proteolytic cleavage. But the product from p20t1 was uncleaved definitely. Conclusions 174S to 201C residues are critical to the cleavage at the LSKGSIVV motif,which might be another essential condition for the cleavage at the LSKGSIVV motif within the conserved SEA module.