May L T, Ghrayeb J, Santhanam U, Tatter S B, Sthoeger Z, Helfgott D C, Chiorazzi N, Grieninger G, Sehgal P B
Rockefeller University, New York, New York 10021.
J Biol Chem. 1988 Jun 5;263(16):7760-6.
The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2). When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity. We have prepared a rabbit polyclonal antiserum to the E. coli-derived human IFN-beta 2 fusion protein. This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations. The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes. "Uninduced" human FS-4 fibroblasts as well as those induced with interleukin-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30). Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by [3H]glucosamine (gp23-gp25). The inclusion of cycloheximide during the [35S]methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet. Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells. Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.
人β2-干扰素（IFN-β2）/B细胞分化因子2/肝细胞刺激因子的互补DNA（cDNA）在大肠杆菌中表达，产生一种融合蛋白，该融合蛋白包含与34个氨基酸的原核前导肽融合的IFN-β2的182个羧基末端氨基酸（rIFN-β2）。当添加到人肝癌细胞系Hep3B2的培养物中时，rIFN-β2以及天然IFN-β2制剂可增强α1-抗糜蛋白酶、补体C3、纤维蛋白原和α1-酸性糖蛋白等阳性急性期反应物的分泌，并抑制白蛋白的分泌，这证实了源自IFN-β2基因的一种蛋白质可具有肝细胞刺激因子活性。我们制备了针对大肠杆菌来源的人IFN-β2融合蛋白的兔多克隆抗血清。这种多克隆抗血清可抑制适当的IFN-β2制剂的肝细胞刺激和B细胞分化活性。抗rIFN-β2抗血清已用于免疫沉淀实验和蛋白质印迹，以帮助确定成纤维细胞和单核细胞中源自IFN-β2基因的分泌蛋白。“未诱导的”人FS-4成纤维细胞以及用白细胞介素-1α、肿瘤坏死因子或细菌脂多糖诱导的细胞分泌至少五种表观分子量在23至30 kDa范围内的IFN-β2形式，这些形式在变性和还原条件下可通过聚丙烯酰胺凝胶电泳分离。当在衣霉素存在下诱导FS-4细胞时，未观察到三种较高分子量形式，这表明这些形式是N-糖基化的（gp28、gp29和gp30）。尽管两种较低分子量形式的分泌对衣霉素有抗性，但它们被[3H]葡糖胺标记（gp23-gp25）。在诱导的FS-4细胞的[35S]甲硫氨酸标记过程中加入环己酰亚胺会导致优先合成和分泌29 kDa的三联体。用细菌脂多糖诱导的人单核细胞也分泌几种大小在23至30 kDa范围内的不同形式的IFN-β2，它们在聚丙烯酰胺凝胶中与从FS-4细胞获得的IFN-β2共迁移。我们的观察结果有助于将先前对多种形式的肝细胞刺激因子的描述与源自IFN-β2基因的特定蛋白质联系起来。
