Funaki T, Fujiwara T, Hong H S, Misumi Y, Nishioka M, Ikehara Y
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Cell Struct Funct. 1996 Feb;21(1):63-72. doi: 10.1247/csf.21.63.
A serum from a patient with HBV hepatitis was found to contain autoantibodies reacting with various mammalian cells. Immunofluorescence staining of cultured cells with the autoantibodies revealed that the antigen was localized at perinuclear regions, where the Golgi markers alpha-mannosidase II and beta-COP were colocalized. The autoantigen disappeared from the perinuclear regions upon incubation with the fungal metabolite brefeldin A, and the immunostainable structures were fragmented into vesicles by treatment with nocodazole. These results strongly indicate that the antigen is localized at the Golgi complex. Immunoblots of cell lysates showed that the autoantibodies recognized a single protein with a molecular mass of 230 kDa in a variety of cell lines, indicating that the 230-kDa antigen is a conserved protein among mammalian species. We designated this protein GCP230 (Golgi complex-associated protein with a molecular mass of 230 kDa). when a postnuclear fraction was prepared and centrifuged, GCP230 was recovered in both cytosol and membrane fractions. Peripheral interaction of GCP230 with membranes was confirmed by phase separation in Triton X-114 solution and by extraction with sodium carbonate. Taken together, these results indicate that GCP230 is a peripheral membrane protein of the Golgi derived from the cytosol, although its function is not known at present.
一名乙肝患者的血清中发现含有能与多种哺乳动物细胞发生反应的自身抗体。用这些自身抗体对培养细胞进行免疫荧光染色显示，抗原定位于核周区域，高尔基标志物α-甘露糖苷酶II和β-COP也共定位于该区域。用真菌代谢产物布雷菲德菌素A孵育后，自身抗原从核周区域消失，用诺考达唑处理后，可免疫染色的结构破碎成小泡。这些结果有力地表明该抗原定位于高尔基体复合体。细胞裂解物的免疫印迹显示，自身抗体在多种细胞系中识别出一种分子量为230 kDa的单一蛋白质，这表明230 kDa的抗原是哺乳动物物种间的保守蛋白。我们将这种蛋白质命名为GCP230（分子量为230 kDa的高尔基体复合体相关蛋白）。制备并离心核后组分时，GCP230在胞质溶胶和膜组分中均被回收。通过在Triton X-114溶液中的相分离以及用碳酸钠提取，证实了GCP230与膜的外周相互作用。综上所述，这些结果表明GCP230是一种源自胞质溶胶的高尔基体外周膜蛋白，尽管其功能目前尚不清楚。
