Malykh Y N, Krisch B, Shaw L, Warner T G, Sinicropi D, Smith R, Chang J, Schauer R
Biochemisches Institut, Christian-Albrechts-Universität zu Kiel, Germany.
Eur J Cell Biol. 2001 Jan;80(1):48-58. doi: 10.1078/0171-9335-00139.
An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.
使用针对CMP-N-乙酰神经氨酸羟化酶的特异性抗体（h-3抗体）和糖缀合物结合的N-羟乙酰神经氨酸（Neu5Gc，这是羟化酶反应的产物）的抗体（α-Gc抗体），对石蜡包埋的猪淋巴结切片进行了免疫组织化学分析。在包括淋巴细胞在内的滤泡周围区细胞中观察到的该酶定位，反映在糖缀合物结合的Neu5Gc的类似分布中。这一结果在组织学水平上证实了先前关于羟化酶在体外调节Neu5Gc生物合成作用的生化研究。对从猪胸腺、脾脏、淋巴结和外周血中分离的淋巴细胞的分析显示，各种淋巴细胞中Neu5Gc的含量存在差异，这与在这些细胞中测定的羟化酶活性密切相关。在外周血淋巴细胞（PBL）中检测到的Neu5Gc含量最高，酶活性也最高。用α-Gc和h-3抗体对石蜡包埋的PBL切片进行免疫组织化学研究表明，这些抗原分别位于细胞表面和细胞质中。使用h-3抗体和免疫金标记的超微结构免疫细胞化学来研究羟化酶的亚细胞定位。该酶在核膜和线粒体外膜附近的细胞质中被检测到，特别是靠近细胞核的线粒体。在细胞质管状结构上也检测到了该抗原。此外，偶尔也观察到高尔基体有微弱的标记。讨论了这种定位可能与底物CMP-Neu5Ac和氧化还原伙伴细胞色素b5的可用性有关的可能性。
