Kleinberg M E, Rotrosen D, Malech H L
Bacterial Diseases Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
J Immunol. 1989 Dec 15;143(12):4152-7.
Cytochrome b558, an essential component of the respiratory burst of phagocytic cells, is the terminal electron donor to molecular oxygen that results in the formation of superoxide anion (O2-.). It is an integral membrane heterodimer that in neutrophils consists of a 22-kDa small subunit and a highly glycosylated 91-kDa large subunit. Identical core proteins often differ in glycosylation in different cell types and with some membrane glycoproteins, the glycosylation state may markedly affect function. In the present study, antisera reactive with cytochrome b558 large subunit was used for immunoblot analysis of the glycosylation pattern of this subunit from different types of phagocytic cells. Striking variability in the apparent m.w. of this broadly banding subunit was detected in five different phagocytic cell types (neutrophils 78,000 to 93,000; eosinophils 74,000 to 115,000; monocytes 82,000 to 99,000; dibutyryl cyclic AMP-induced HL-60 cells 79,000 to 103,000; dimethyl sulfoxide-induced HL-60 cells 77,000 to 110,000). However, after complete cleavage of N-linked oligosaccharides with endoglycosidase F, the core peptide of cytochrome b558 large subunit from these different cell types had the same Mr (58,000). Inhibition of N-glycosylation with tunicamycin in differentiating HL-60 cells resulted in the synthesis of immunoreactive protein of the same m.w. and banding pattern as seen after endoglycosidase F cleavage. These tunicamycin treated cells retained some capacity to generate superoxide anion when stimulated with PMA. We conclude that the identity of the N-linked oligosaccharides of the cytochrome b558 large subunit differ in various phagocytic cells. All N-linked glycans on cytochrome b558 in all cell types examined were of the complex type as defined by resistance to endoglycosidase H cleavage. N-linked glycosylation of the cytochrome b558 large subunit may not be essential for activation of the respiratory burst.
细胞色素b558是吞噬细胞呼吸爆发的重要组成部分，是分子氧的末端电子供体，可导致超氧阴离子（O2-）的形成。它是一种整合膜异二聚体，在中性粒细胞中由一个22 kDa的小亚基和一个高度糖基化的91 kDa大亚基组成。相同的核心蛋白在不同细胞类型中的糖基化情况往往不同，对于一些膜糖蛋白而言，糖基化状态可能会显著影响其功能。在本研究中，使用与细胞色素b558大亚基反应的抗血清对来自不同类型吞噬细胞的该亚基的糖基化模式进行免疫印迹分析。在五种不同的吞噬细胞类型（中性粒细胞78,000至93,000；嗜酸性粒细胞74,0至115,000；单核细胞82,000至99,000；二丁酰环磷酸腺苷诱导的HL-60细胞79,000至103,000；二甲亚砜诱导的HL-60细胞77,000至110,000）中检测到该宽条带亚基的表观分子量存在显著差异。然而，用内切糖苷酶F完全切割N-连接寡糖后，来自这些不同细胞类型的细胞色素b5于8大亚基的核心肽具有相同的Mr（58,000）。在分化的HL-60细胞中用衣霉素抑制N-糖基化导致合成的免疫反应性蛋白具有与内切糖苷酶F切割后相同的分子量和条带模式。这些经衣霉素处理的细胞在用佛波酯刺激时仍保留一定产生超氧阴离子的能力。我们得出结论，细胞色素b558大亚基的N-连接寡糖在各种吞噬细胞中不同。在所检查的所有细胞类型中，细胞色素b558上的所有N-连接聚糖均为复杂型，这是通过对内切糖苷酶H切割的抗性来定义的。细胞色素b558大亚基的N-连接糖基化对于呼吸爆发的激活可能不是必需的。
