Imber M J, Pizzo S V
J Biol Chem. 1981 Aug 10;256(15):8134-9.
These studies explore the role of conformational change and exposed carbohydrate residues in the clearance of alpha 2-macroglobulin-trypsin (alpha 2M-T) complexes in the mouse. Human alpha 2-macroglobulin (alpha 2M) was purified and demonstrated to be homogeneous in the electrophoretic "slow" form. Two conformationally altered derivatives, alpha 2M-T and alpha 2-macroglobulin-methylamine (alpha 2M-MeNH2), were prepared and demonstrated to exist in the electrophoretic "fast" form. Radiolabeled alpha 2M-T and alpha 2M-MeNH2 were cleared rapidly with a half-life of 2-4 min following injection into mice. Radiolabeled native alpha 2M, however, remained in the circulation with a half-life of several hours. Both alpha 2M-T and alpha 2M-MeNH2 bound specifically to mouse peritoneal macrophages at 4 degrees C and occupancy of receptor sites increased with increasing time and radioligand concentration. Excess amounts of unlabeled alpha 2M-T or alpha 2M-MeNH2 cross-completed with trace amounts of the other in both clearance studies and binding assays, indicating that both derivatives were removed by the same receptor pathway. The clearance and binding of alpha 2M-T and alpha 2M-MeNH2 were not inhibited by excess amounts of unlabeled asialoorosomucoid, fucosyl-bovine serum albumin, mannosyl-BSA, or N-acetylglucosaminyl-BSA. Our results indicate that the clearance pathway removing alpha 2M-T complexes from the circulation recognizes a fundamental conformational change in alpha 2M secondary to protease binding, which can also be induced by exposure to methylamine. Therefore, other chemical or physical alterations that occur in alpha 2M upon binding trypsin, apart from the conformational change also present in alpha 2M-MeNH2, do not seem necessary for the recognition of alpha 2M-T by cells in the clearance pathway. In addition, this pathway appears distinct from several systems already described mediating clearance of glycoproteins through recognition of terminal galactose, fucose, N-acetylglucosamine, or mannose on oligosaccharide side chains.
这些研究探讨了构象变化和暴露的碳水化合物残基在小鼠体内α2-巨球蛋白-胰蛋白酶（α2M-T）复合物清除过程中的作用。人α2-巨球蛋白（α2M）被纯化，并证明在电泳“慢”形式下是均匀的。制备了两种构象改变的衍生物，α2M-T和α2-巨球蛋白-甲胺（α2M-MeNH2），并证明它们以电泳“快”形式存在。将放射性标记的α2M-T和α2M-MeNH2注射到小鼠体内后，它们迅速被清除，半衰期为2-4分钟。然而，放射性标记的天然α2M在循环中保留数小时的半衰期。α2M-T和α2M-MeNH2在4℃时都特异性地结合到小鼠腹膜巨噬细胞上，并且随着时间和放射性配体浓度的增加，受体位点的占有率增加。在清除研究和结合试验中，过量的未标记α2M-T或α2M-MeNH2与微量的另一种物质相互竞争，这表明两种衍生物都通过相同的受体途径被清除。α2M-T和α2M-MeNH2的清除和结合不受过量未标记的去唾液酸糖蛋白、岩藻糖基-牛血清白蛋白、甘露糖基-BSA或N-乙酰葡糖胺基-BSA的抑制。我们的结果表明，从循环中清除α2M-T复合物的途径识别了α2M因蛋白酶结合而发生的基本构象变化，这种变化也可由暴露于甲胺诱导。因此，除了α2M-MeNH2中也存在的构象变化外，α2M与胰蛋白酶结合时发生的其他化学或物理改变，对于清除途径中细胞识别α2M-T似乎并非必要。此外，该途径似乎不同于已经描述的几种通过识别寡糖侧链上的末端半乳糖、岩藻糖、N-乙酰葡糖胺或甘露糖来介导糖蛋白清除的系统。
