Yao C, Jasmer D P
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman 99164-7040, USA.
Mol Biochem Parasitol. 1998 May 1;92(2):207-18. doi: 10.1016/s0166-6851(97)00199-0.
Infection of mammalian skeletal muscle cells by Trichinella spiralis induces a series of changes that include: reentry of the terminally differentiated host cell into the cell cycle; suspension of infected cells in apparent G2/M; and transcriptional inactivation of the differentiated skeletal muscle gene program. Cell cycle repositioning and genetic reprogramming are chronic characteristics of host cells that can remain infected for years. Nuclear antigens (NA, 79, 86 and 97 kDa) that localize to host cell nuclei have been detected with antibodies against T. spiralis proteins. Since NA may play a role in regulating the infected cell phenotype, their origin, nuclear compartmentalization, and biochemical properties were investigated. We show that a monoclonal antibody to a defined epitope of T. spiralis glycans binds these NA, which indicates the parasite origin of these proteins. NA were not extracted under conditions that solubilized chromatin from infected cell nuclei. In contrast, NA were coextracted with B lamins (nuclear envelope) by 4 M urea. Urea extraction was pH dependent (8.0), suggesting ionic interaction of NA in protein complexes. Nevertheless, confocal microscopy demonstrated colocalization of NA with host chromatin, and not B lamins. Nuclear protein complexes containing NA were observed under non-reducing conditions, and NA were readily cross-linked in isolated nuclei by succinimidyl protein conjugating reagents. The results establish methods to extract NA from infected cell nuclei for further biochemical analysis, establish the existence of nuclear protein complexes containing NA and demonstrate colocalization of NA with host chromatin. Collectively, the results provide a foundation from which to investigate the role of NA in regulating the T. spiralis infected skeletal muscle cell phenotype.
旋毛虫感染哺乳动物骨骼肌细胞会引发一系列变化，包括：终末分化的宿主细胞重新进入细胞周期；感染细胞停滞在明显的G2/M期；以及分化的骨骼肌基因程序的转录失活。细胞周期重新定位和基因重编程是宿主细胞的慢性特征，这些细胞可能会持续感染数年。利用抗旋毛虫蛋白的抗体检测到定位于宿主细胞核的核抗原（NA，分子量分别为79、86和97 kDa）。由于NA可能在调节感染细胞表型中发挥作用，因此对它们的起源、核区室化和生化特性进行了研究。我们发现，一种针对旋毛虫聚糖特定表位的单克隆抗体可结合这些NA，这表明这些蛋白源自寄生虫。在能溶解感染细胞核染色质的条件下，NA无法被提取出来。相反，NA可通过4 M尿素与B型核纤层蛋白（核膜）共同提取。尿素提取依赖于pH值（8.0），这表明NA在蛋白质复合物中存在离子相互作用。然而，共聚焦显微镜显示NA与宿主染色质共定位，而非与B型核纤层蛋白共定位。在非还原条件下观察到含有NA的核蛋白复合物，并且NA在分离的细胞核中很容易被琥珀酰亚胺基蛋白偶联试剂交联。这些结果建立了从感染细胞核中提取NA以进行进一步生化分析的方法，证实了含有NA的核蛋白复合物的存在，并证明了NA与宿主染色质的共定位。总体而言，这些结果为研究NA在调节旋毛虫感染的骨骼肌细胞表型中的作用奠定了基础。
