Veugelers Kirstin, Motyka Bruce, Goping Ing Swie, Shostak Irene, Sawchuk Tracy, Bleackley R Chris
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Mol Biol Cell. 2006 Feb;17(2):623-33. doi: 10.1091/mbc.e05-07-0631. Epub 2005 Nov 9.
During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate.
在细胞毒性T淋巴细胞或自然杀伤细胞通过颗粒介导的杀伤过程中，丝氨酸蛋白酶颗粒酶B通过内吞作用进入靶细胞并诱导细胞凋亡。先前的研究表明甘露糖6-磷酸受体发挥了作用，但对纯化的颗粒酶B进行的进一步实验表明这并非必不可少。此外，现在清楚的是，颗粒酶B与蛋白聚糖丝甘氨酸以高分子量复合物的形式从杀伤细胞中胞吐出来。在此，颗粒酶B以纯化的单体形式递送，或与糖胺聚糖或丝甘氨酸形成复合物递送，并对杀伤作用进行评估。当颗粒酶B为单体时，可溶性甘露糖6-磷酸的影响有限，而与颗粒酶B形成复合物诱导的细胞凋亡则被甘露糖6-磷酸有效抑制。最重要的是，当颗粒酶B和穿孔素从颗粒中一起递送时，也观察到了甘露糖6-磷酸的抑制作用。在由不依赖阳离子的甘露糖6-磷酸受体介导的下拉实验中，在固定化硫酸乙酰肝素存在的情况下，颗粒酶B与该受体的结合更强烈。因此，我们提出了一个模型，即在生理条件下，与丝甘氨酸结合的颗粒酶B通过甘露糖6-磷酸受体进行关键的内吞作用，并且细胞表面的硫酸乙酰肝素会增强受体结合。
