Weber S, Babina M, Hermann B, Henz B M
Department of Dermatology, Virchow Clinics, Humboldt-Universität zu Berlin, Germany.
Immunobiology. 1997 Jun;197(1):82-96. doi: 10.1016/S0171-2985(97)80059-9.
Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA). The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA. Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA. In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6). PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation. The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane. Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43. In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors. Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant. Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced. In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells. Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells.
白细胞唾液酸蛋白（CD43）是循环白细胞上的主要唾液酸蛋白，先前已有报道称，在用佛波酯肉豆蔻酸乙酸酯（PMA）激活后，中性粒细胞上的该蛋白表达下调。之前检测的其他单细胞，即血液淋巴细胞，在受到PMA刺激时不会下调CD43的表达。最近，我们对人肥大细胞系HMC - 1上的白细胞唾液酸蛋白进行了特性分析，发现用PMA刺激后白细胞唾液酸蛋白表达下调。在本研究中，我们探讨了PMA介导的HMC - 1细胞（亚克隆5C6）上CD43下调的机制。PMA在HMC - 1细胞激活过程中导致可溶性白细胞唾液酸蛋白（123 kD）的释放。可溶性白细胞唾液酸蛋白的分子量与细胞膜结合分子的分子量几乎相同，这表明是在靠近细胞膜处发生了裂解。丝氨酸蛋白酶抑制剂，如苯甲基磺酰氟（PMSF）、苯甲脒和3,4 - 二氯异香豆素，可阻断PMA介导的CD43裂解。在所有实验中，对CD43下调的抑制作用取决于蛋白酶抑制剂的浓度。用各种蛋白酶（胰蛋白酶、α - 糜蛋白酶、弹性蛋白酶、木瓜蛋白酶、纳豆酶）处理HMC - 1细胞会显著降低抗CD43的结合能力，并导致可溶性白细胞唾液酸蛋白（116 kD）或其片段释放到上清液中。用霍乱弧菌或脲节杆菌的神经氨酸酶预处理HMC - 1细胞会导致CD43对蛋白酶的敏感性增加，而PMA的作用不受影响。总之，首次在中性粒细胞以外的细胞，即HMC - 1细胞中描述了CD43的蛋白水解裂解。我们的结果表明，丝氨酸蛋白酶参与了PMA介导的HMC - 1细胞上白细胞唾液酸蛋白的下调过程。
