Perez-Vilar Juan, Randell Scott H, Boucher Richard C
Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Glycobiology. 2004 Apr;14(4):325-37. doi: 10.1093/glycob/cwh041. Epub 2004 Jan 12.
We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.
我们在COS-7细胞中表达重组半胱氨酸亚结构域，以研究这个高度保守的蛋白质结构域在粘蛋白生物合成中的作用。分别在MUC5AC和MUC5B中表达完整的Cys1和Cys5以及Cys1和Cys3亚结构域，每个亚结构域都带有六个羧基末端组氨酸残基，用[³⁵S]半胱氨酸/甲硫氨酸进行脉冲标记，然后在培养基中检测标记的蛋白质。在非还原条件下，分泌的半胱氨酸亚结构域为单体，表明不存在链间二硫键。交联研究表明这些结构域能够通过非常弱的非共价相互作用相互作用。尽管这些结构域的表观分子量与缺乏N-和O-聚糖一致，但它们可以用甘露糖特异性凝集素纯化。假定的C-甘露糖基化受体基序WXXW中的第一个色氨酸残基发生突变会阻止凝集素结合，这表明C-甘露糖基化负责凝集素结合。通过脉冲追踪实验判断，C-甘露糖基化在结构域生物合成过程中很早就发生了，可能在内质网（ER）中。WXXW基序发生突变或在C-甘露糖基化缺陷细胞系CHO-Lec35.1细胞中表达未突变的结构域，会导致相应半胱氨酸亚结构域的分泌减少。与融合到半胱氨酸亚结构域的绿色荧光蛋白在CHO-K1细胞中表达的相同结构域相比，活细胞成像清楚地显示CHO-Lec35.1细胞内质网中半胱氨酸亚结构域的存在增加。综合考虑，这些研究表明MUC5AC和MUC5B的半胱氨酸亚结构域在其各自的WXXW基序中被C-甘露糖基化。C-甘露糖基化可能是半胱氨酸亚结构域正确折叠和/或粘蛋白生物合成过程中内质网输出的某些方面所必需的。
