Lillehoj Erik P, Han Feng, Kim K Chul
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 N Pine St, Baltimore, MD 21201, USA.
Biochem Biophys Res Commun. 2003 Aug 1;307(3):743-9. doi: 10.1016/s0006-291x(03)01260-9.
MUC1 mucin is a type 1 transmembrane glycoprotein dimer of extracellular and membrane-bound subunits. The two non-covalently associated subunits are produced from a single polypeptide chain by proteolysis at a Gly-Ser peptide bond in the endoplasmic reticulum prior to localization on the cell surface. However, once expressed on the surface, the extracellular subunit is shed from cells in the absence of the membrane-associated subunit. Previous studies implicated a cellular metalloproteinase mediating MUC1 ectodomain shedding, but no reports have delineated the site of metalloproteinase cleavage or directly assessed the role of the Gly-Ser bond in shedding. Therefore, we performed site-directed mutagenesis of the Gly-Ser site and determined the effects on MUC1 proteolysis and shedding. Ser-->Ala substitution blocked MUC1 cleavage and inhibited shedding. Equal amounts of wild type and mutant MUC1 were expressed on the cell surface, indicating that lack of shedding of the mutant molecule was not due to reduced surface localization. We conclude that the Gly-Ser peptide bond is required for MUC1 shedding.
黏蛋白1（MUC1）是一种由细胞外亚基和膜结合亚基组成的1型跨膜糖蛋白二聚体。这两个非共价结合的亚基是在内质网中，由一条多肽链在定位到细胞表面之前，通过在甘氨酸 - 丝氨酸肽键处的蛋白水解作用产生的。然而，一旦在表面表达，细胞外亚基会在没有膜相关亚基的情况下从细胞上脱落。先前的研究表明有一种细胞金属蛋白酶介导MUC1胞外域的脱落，但尚无报道描述金属蛋白酶切割的位点，或直接评估甘氨酸 - 丝氨酸肽键在脱落中的作用。因此，我们对甘氨酸-丝氨酸位点进行了定点诱变，并确定了其对MUC1蛋白水解和脱落的影响。丝氨酸替换为丙氨酸阻断了MUC1的切割并抑制了脱落。等量的野生型和突变型MUC1在细胞表面表达，表明突变分子的脱落缺失不是由于表面定位减少所致。我们得出结论，甘氨酸 - 丝氨酸肽键是MUC1脱落所必需的。
