Kiyohara T, Dennis J W, Boegman R J, Roder J C
J Immunol. 1985 Jul;135(1):659-64.
Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.
糖苷酶被用作探针来分析自然杀伤（NK）细胞介导的细胞毒性机制。用一种外切糖苷酶α-甘露糖苷酶预处理经尼龙毛富集的CBA/J脾细胞、一种小鼠NK克隆或人外周血淋巴细胞（PBL），导致对YAC-1.2或K562肿瘤细胞的NK裂解活性出现明显的剂量依赖性抑制。在37℃对小鼠效应细胞进行60分钟预处理后出现最大抑制，且α-甘露糖苷酶对NK抑制的动力学与报道的酶活性动力学相似。在用抑制NK剂量的α-甘露糖苷酶处理的小鼠脾细胞上清液中通过化学方法检测到释放的己糖，用乙二胺四乙酸（EDTA）使酶功能失活可逆转NK抑制作用。这些结果表明α-甘露糖苷酶凭借其酶促作用抑制NK功能。用该酶处理后人PBL培养20小时导致NK裂解功能恢复超过70%。通过在培养基中加入衣霉素（一种天冬酰胺连接糖蛋白的糖基化抑制剂）可阻断恢复。这些结果表明α-甘露糖苷酶敏感位点可能是重新合成的糖蛋白。神经氨酸酶、β-半乳糖苷酶、内切β-N-乙酰葡糖胺酶-D和H以及肽-N-糖苷酶处理均未抑制人NK细胞对K562细胞的裂解。用α-甘露糖苷酶预处理经尼龙毛富集的CBA/J脾细胞或经Percoll富集的人大颗粒淋巴细胞（LGL），分别不影响它们与YAC 1.2靶细胞或K562靶细胞结合的能力。钙离子脉冲实验表明，NK细胞上的α-甘露糖苷酶敏感位点在靶细胞与效应细胞结合后但在钙离子内流之前起作用。用该酶对效应细胞进行预处理通常发生在效应细胞与靶细胞相互作用之后。这些结果表明磷脂甲基化反应与NK细胞上的α-甘露糖苷酶敏感位点相关联。类似于其他生理系统，如肥大细胞中组胺的释放，NK细胞中磷脂甲基化的触发可能作为跨质膜信号转导的一种机制。
