Nan Xinli, Carubelli Ivan, Stamatos Nicholas M
Institute of Human Biology, and Department of Medicine, University of Maryland Medical Center, 725 West Lombard Street, Baltimore, MD 21201, USA.
J Leukoc Biol. 2007 Jan;81(1):284-96. doi: 10.1189/jlb.1105692. Epub 2006 Oct 6.
Sialidases influence cellular activity by removing terminal sialic acid from glycoproteins and glycolipids. Four genetically distinct sialidases (Neu1-4) have been identified in mammalian cells. In this study, we demonstrate that only lysosomal Neu1 and plasma membrane-associated Neu3 are detected in freshly isolated and activated human T lymphocytes. Activation of lymphocytes by exposure to anti-CD3 and anti-CD28 IgG resulted in a ninefold increase in Neu1-specific activity after growth of cells in culture for 5 days. In contrast, the activity of Neu3 changed minimally in activated lymphocytes. The increase in Neu1 enzyme activity correlated with increased synthesis of Neu1-specific mRNA. Neu1 was present on the surface of freshly isolated and activated CD4 and CD8 T lymphocytes, as determined by staining intact cells with anti-Neu1 IgG and analysis by flow cytometry and by Western blot analysis of biotin-labeled cell surface proteins. Cell surface Neu1 was found tightly associated with a subunit of protective protein/cathepsin A (PPCA). Compared with freshly isolated lymphocytes, activated cells expressed more surface binding sites for galactose-recognizing lectins Erythrina cristagalli (ECA) and Arachis hypogaea. Growth of cells in the presence of sialidase inhibitors 2,3-dehydro-2-deoxy-N-acetylneuraminic acid or 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid resulted in a smaller increase in number of ECA-binding sites and a greater amount of cell surface sialic acid in activated cells. Inhibition of sialidase activity also resulted in reduced expression of IFN-gamma in activated cells. The down-regulation of IFN-gamma occurred at the transcriptional level. Thus, sialidase activity in activated T lymphocytes contributes to the hyposialylation of specific cell surface glycoconjugates and to the production of IFN-gamma.
唾液酸酶通过去除糖蛋白和糖脂上的末端唾液酸来影响细胞活性。在哺乳动物细胞中已鉴定出四种基因不同的唾液酸酶（Neu1 - 4）。在本研究中，我们证明在新鲜分离和活化的人T淋巴细胞中仅检测到溶酶体Neu1和与质膜相关的Neu3。用抗CD3和抗CD28 IgG刺激淋巴细胞活化，细胞在培养5天后，Neu1特异性活性增加了九倍。相比之下，活化淋巴细胞中Neu3的活性变化极小。Neu1酶活性的增加与Neu1特异性mRNA合成的增加相关。通过用抗Neu1 IgG对完整细胞进行染色、流式细胞术分析以及对生物素标记的细胞表面蛋白进行蛋白质印迹分析确定，Neu1存在于新鲜分离和活化的CD4和CD8 T淋巴细胞表面。发现细胞表面的Neu1与保护蛋白/组织蛋白酶A（PPCA）的一个亚基紧密相关。与新鲜分离的淋巴细胞相比，活化细胞对识别半乳糖的凝集素刺桐（ECA）和花生（Arachis hypogaea）表达更多的表面结合位点。在唾液酸酶抑制剂2,3 - 脱氢 - 2 - 脱氧 - N - 乙酰神经氨酸或4 - 胍基 - 2 - 脱氧 - 2,3 - 脱氢 - N - 乙酰神经氨酸存在下培养细胞，导致活化细胞中ECA结合位点数量的增加幅度较小，细胞表面唾液酸含量较多。唾液酸酶活性的抑制还导致活化细胞中IFN - γ的表达降低。IFN - γ的下调发生在转录水平。因此，活化T淋巴细胞中的唾液酸酶活性有助于特定细胞表面糖缀合物的低唾液酸化以及IFN - γ的产生。
