摘要
目的探讨大鼠黏蛋白3(Muc3)羧基端SEA组件的完整性与蛋白酶切的关系。方法对截断了的大鼠Muc3的羧基端(p20t和p20SEA)采用定点突变的方法在所要求的位点插入终止密码子,采用SDS/PAGE和Western blot方法检测蛋白表达,N-glycosidase F消化法对表达蛋白质行脱N型糖链。结果Muc3完整的羧基端产物翻译后经蛋白酶切产生30kD的氨基端酶切片段和49kD的羧基端酶切片段,30kD的氨基端酶切片段经脱N型糖链后分子量变为22kD;含完整SEA组件但不含SEA组件后续氨基酸的截断大鼠Muc3羧基端用V5抗体可以检测到30kD的表达产物,脱N型糖链后分子量变为22kD;含不完整SEA组件的截断的大鼠Muc3羧基端用V5抗体可以检测到26～30kD的表达产物,脱N型糖链后分子量变为26kD。结论Muc3完整的羧基端产物和SEA组件完整但不含SEA组件后续氨基酸的大鼠Muc3羧基端均在翻译后经历了蛋白酶切,但是SEA组件不完整的大鼠Muc3羧基端在翻译后未能出现蛋白酶切,提示SEA组件完整性是Muc3羧基端蛋白酶切反应发生的一个重要条件。
Objective It has been known that the C terminal domain of rodent Muc3 underwent proteolytic digestion. G/S within the motif of cleavage, LSKGSIVV, was one of the important pivots for digestion. The present investigation was aiming at exploring the unknown relationship between the integrity of SEA module and the proteolytic digestion. Method Truncated rodent Muc3 C-terminal do- mains （p20t and p20SEA） were produced by site-directed mutagenesis to insert a stop code in the required place. Proteins were detected by SDS/PAGE and Western blotting. Deglycosylation of the expressed protein was performed by digestion using N glycosidase F. Results Muc3 C-terminal domain was posttranslationally cleaved to produce a V5 tagged 30kDa extracellular glycopeptide and a Myc-tagged 49kDa membrane-associated glycopeptide. The 30kDa N-terminal fragment shifted to 22kDa after deglycosylation. The truncated rodent Muc3 C-terminal domain containing complete SEA module, but without the following residues after SEA module, was 30kDa Mw as detected with anti-V5 antibody, and it was shifted to 22kDa after deglycosylation. But the truncated rodent Muc3 C-terminal domain containing incomplete SEA module （p20t） of 26-30kDa Mw was shifted to 26kDa after deglycosylation. Conclusion There was proteolytic digestion in both complete rodem C-terminal domain and complete SEA module without residues after SEA module. But proteolytic digestion does not occur in the incomplete SEA module of rodent Muc3. So it may be concluded that the integrity of SEA module of rodent Muc3 was also a crucial condition for its proteolytic digestion.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第7期726-728,共3页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助项目(30300121
30470401)
教育部归国人员启动基金项目
第三军医大学启动基金资助项目
