黏蛋白3羧基末端蛋白酶切的阻断及N-糖基化的抑制对其细胞膜定位的影响

Influence of proteolytic cleavage blockage and N-linked oligosaccharide inhibition on the membrane targeting of rodent Muc3 C-terminal domain

目的探讨大鼠黏蛋白3(rMuc3)羧基端蛋白酶切反应和羧基端内N-糖苷型糖链与细胞膜定位的关系。方法利用脂质体将包含rMuc3羧基端的3个真核表达载体p20、p20t和p20s/a导入COS-1细胞,并采用N-糖苷型糖链合成抑制剂衣霉素处理转染后的COS-1细胞,抑制其表达蛋白质的N型糖链合成。采用免疫荧光定位法,用抗V5抗体和抗Myc抗体检测Muc3蛋白的表达。结果在甲醇固定的COS-1细胞,细胞核周和细胞表面均能检测到完整的Muc3羧基端p20表达产物;在未经甲醇固定的COS-1细胞,免疫荧光仅限于细胞表面。缺失的rMuc3羧基端的p20t表达产物仅能在细胞核周检测到。Muc3羧基端建立的蛋白酶切阴性突变体p20s/a与p20转染细胞的荧光分布相近。衣霉素抑制糖链合成后不影响膜定位。结论Muc3羧基末端蛋白酶切的阻断及N-糖基化的抑制不能影响Muc3的细胞膜定位,影响其定位的因素主要是SEA组件靠近羧基端的区域,包括该分子的跨膜区和胞质内区域。

Objective To explore the correlation between membrane targeting of rodent Muc3 C-terminal domain and proteolytic cleavage blockage within its SEA module and N-linked oligosaccharides inhibition.Methods COS-1 cells were transfected with three different expression vectors containing rodent Muc3 C-terminal domain,namely p20,p20t and p20s/a by lipofectAMINE reagent.Inhibition of N-glycosylation of the expressed protein was performed by using tunicamycin.The transfected COS-1 cells（fixed or unfixed） were detected by immunolocalization experiments（anti-V5 and anti-Myc antibody） for the protein expression.Results In fixed COS-1 cells,the expressed product of p20 transfectant detected using both anti-Myc and anti-V5 antibodies was found to localize in perinuclear position and on the plasma membrane.While in the unfixed cells,immunostaining was only confined on cell surface using anti-V5 antibody.The expressed product of p20t transfectant was detected by anti-V5 antibody to localize only in perinuclear region,as observed in a few fixed cells.The distribution of p20s/a fluorescence resembled that of p20 transfectant.Plasma membrane targeting of the non-glycosylated products due to tunicamycin treatment still occurred in transfected COS-1 cells and resembled the glycosylated products.Conclusions The blockage of proteolytic cleavage within C-terminal domain of rodent Muc3 and its inhibition of N-linked oligosaccharides in SEA module cannot affect its membrane targeting.The only apparent requirement for membrane targeting is the transmembrane and/or cytoplasmic tail segments which exist in the C-terminal domains of rMuc3.