Seelig H P, Schranz P, Schröter H, Wiemann C, Griffiths G, Renz M
Institute of Immunology and Molecular Genetics, Karlsruhe, Germany.
Mol Cell Biol. 1994 Apr;14(4):2564-76. doi: 10.1128/mcb.14.4.2564-2576.1994.
Molecular genetic analyses of a 376-kDa Golgi complex (GC) membrane protein (giantin) are described. The immunoglobulin G fraction of a human serum containing antibodies against GC antigens as revealed by indirect immunofluorescence microscopy with Hep-2 cells was used to screen a HeLa cDNA expression library, yielding four overlapping cross-hybridizing clones. Additional cDNA clones were retrieved from a lambda gt11 human thyroid cDNA library or generated by reverse transcriptase-mediated PCR from HeLa cell mRNA. Alignment of the clones resulted in a consensus cDNA of 10,300 bp encoding a protein of 376 kDa. The corresponding mRNA with a size of about 10 kb was detected by Northern (RNA) blotting of HeLa, Hep-G2, and Jurkat cell RNA. Sequence analyses of the protein revealed an extraordinarily high content of heptad repeats with the probability of forming coiled coils similar to the proteins of the myosin family. Five overlapping recombinant proteins covering the entire sequence were synthesized and used for antibody production in rabbits and for affinity purification of human and rabbit antibodies. Indirect immunofluorescence experiments also done with brefeldin A-treated Hep-2 and Pt K1 cells revealed an identical GC staining of both the affinity-purified human and rabbit antibodies. Double labeling experiments with antibodies against the GC marker mannosidase II as well as immunoelectron microscopic studies confirmed the localization of the protein within the GC. A corresponding endogenous large-molecular-mass protein of about 390 kDa was found in [35S]methionine-labeled Hep-2 cell lysates as well as in GC-enriched subcellular fractions from rat liver. The protein as well as the recently described proteins golgin-95 and golgin-160 (M. J. Fritzler, J. C. Hamel, R. L. Ochs, and E. K. L. Chan, J. Exp. Med. 178:49-62, 1993) may belong to a new group of Golgi proteins with a high content of heptad repeats which may exert functions in scaffold formation or vesicle transport. As far as can be concluded from immunological and personally communicated partial cDNA sequence data, the protein seems to be identical with a 400-kDa Golgi protein (giantin) recently described (A. D. Linstedt and H. P. Hauri, Mol. Biol. Cell 4:679-693, 1993). Therefore, we agreed to adopt the name giantin.
本文描述了对一种376 kDa高尔基体复合物（GC）膜蛋白（巨蛋白）的分子遗传学分析。用人血清的免疫球蛋白G组分（该血清含有针对GC抗原的抗体，通过用Hep - 2细胞进行间接免疫荧光显微镜检查得以揭示）筛选HeLa cDNA表达文库，得到四个重叠的交叉杂交克隆。另外的cDNA克隆是从λgt11人甲状腺cDNA文库中获取的，或者通过逆转录酶介导的PCR从HeLa细胞mRNA生成。对这些克隆进行比对，得到一个10300 bp的共有cDNA，其编码一个376 kDa的蛋白质。通过对HeLa、Hep - G2和Jurkat细胞RNA进行Northern（RNA）印迹分析，检测到大小约为10 kb的相应mRNA。对该蛋白质的序列分析显示其七肽重复序列含量极高，有可能形成类似于肌球蛋白家族蛋白质的卷曲螺旋结构。合成了覆盖整个序列的五个重叠重组蛋白，并用于在兔体内产生抗体以及亲和纯化人和兔的抗体。用布雷菲德菌素A处理过的Hep - 2和Pt K1细胞进行的间接免疫荧光实验也表明，亲和纯化的人和兔抗体对GC的染色相同。用针对GC标志物甘露糖苷酶II的抗体进行的双重标记实验以及免疫电子显微镜研究证实了该蛋白质在GC内的定位。在[35S]甲硫氨酸标记的Hep - 2细胞裂解物以及大鼠肝脏富含GC的亚细胞组分中，发现了一种相应的约390 kDa的内源性大分子质量蛋白质。该蛋白质以及最近描述的蛋白质golgin - 95和golgin - 160（M. J. Fritzler、J. C. Hamel、R. L. Ochs和E. K. L. Chan，《实验医学杂志》178:49 - 62，1993）可能属于一组新的高尔基体蛋白质，其七肽重复序列含量高，可能在支架形成或囊泡运输中发挥作用。根据免疫学和个人交流的部分cDNA序列数据可以推断，该蛋白质似乎与最近描述的一种400 kDa高尔基体蛋白质（巨蛋白）相同（A. D. Linstedt和H. P. Hauri，《分子生物学细胞》4:679 - 693，1993）。因此，我们同意采用巨蛋白这个名称。
