Hanley J M, Haugen T H, Heath E C
J Biol Chem. 1983 Jun 25;258(12):7858-69.
Native rat haptoglobin is an heterotetramer consisting of two alpha-subunits (Mr = approximately 9,500) and two glycosylated beta-subunits (Mr = approximately 38,000) joined by interchain disulfide bonds. We previously reported (Haugen, T. H., Hanley, J. M., and Heath, E. C. (1981) J. Biol. Chem. 256, 1055-1057) that the synthesis of rat haptoglobin is encoded by a single mRNA, and that the primary in vitro translation product is a single polypeptide, preprohaptoglobin (Mr = approximately 40,000), that contains an NH2-terminal signal sequence as well as an alpha-subunit region and a beta-subunit region. We now report that partial sequence analysis of preprohaptoglobin indicates that the protein possesses an NH2-terminal hydrophobic signal peptide of 18 amino acid residues, followed directly by the alpha-subunit region, with the beta-subunit region located in the carboxyl-terminal portion of the protein. The co-translationally processed translation product consists of a core glycosylated polypeptide, prohaptoglobin (Mr = approximately 45,000), that is devoid of the signal sequence and possesses both the alpha-subunit and beta-subunit regions of haptoglobin. Pulse-chase experiments in cultures of isolated hepatocytes, and analysis of haptoglobin biosynthetic intermediates in the various subcellular organelles of in vivo labeled rat liver indicate that: (a) in the endoplasmic reticulum, core glycosylated prohaptoglobin is dimerized and a portion of the protein is processed to form the individual alpha- and beta-subunits; (b) the carbohydrate side chains of prohaptoglobin and of core glycosylated beta-subunit (Mr = approximately 35,000) are converted to complex, sialylated side chains in the Golgi apparatus, resulting in the formation of fully glycosylated prohaptoglobin (Mr = approximately 48,000) and beta-subunit (Mr = approximately 38,000), and these forms of the protein, as well as the alpha-subunit (Mr = approximately 9,500), are secreted; (c) inhibition of glycosylation with tunicamycin does not significantly affect the rate of synthesis, processing, or secretion of the various haptoglobin polypeptides in isolated hepatocytes; (d) similar experiments conducted in the presence of colchicine also had no effect on the rate of synthesis and processing of the intermediates; and (e) the species of haptoglobin secreted in vivo and from isolated hepatocytes consist of approximately 60-70% in the form of the alpha 2 beta 2 tetramer, and the remainder as dimerized prohaptoglobin. Presumably, secreted prohaptoglobin may be processed to the native subunit structure after secretion, as we demonstrated that incubation of prohaptoglobin with either normal rat serum, rat plasma, or with the sera of other animal species results in its conversion to the corresponding alpha- and beta-subunits of the native protein.
天然大鼠触珠蛋白是一种异源四聚体，由两条α亚基（Mr = 约9500）和两条糖基化的β亚基（Mr = 约38000）通过链间二硫键连接而成。我们之前报道过（豪根，T. H.，汉利，J. M.，和希思，E. C.（1981年）《生物化学杂志》256卷，1055 - 1057页），大鼠触珠蛋白的合成由单一mRNA编码，并且体外翻译的初级产物是单一多肽，即前原触珠蛋白（Mr = 约40000），它含有一个NH2端信号序列以及一个α亚基区域和一个β亚基区域。我们现在报道，前原触珠蛋白的部分序列分析表明，该蛋白具有一个由18个氨基酸残基组成的NH2端疏水信号肽，紧接着是α亚基区域，β亚基区域位于蛋白的羧基末端部分。共翻译加工后的翻译产物是一种核心糖基化多肽，即原触珠蛋白（Mr = 约45000），它没有信号序列，并且具有触珠蛋白的α亚基和β亚基区域。在分离的肝细胞培养物中进行的脉冲追踪实验，以及对体内标记的大鼠肝脏各种亚细胞器中触珠蛋白生物合成中间体的分析表明：（a）在内质网中，核心糖基化的原触珠蛋白二聚化，并且一部分蛋白被加工形成单个的α和β亚基；（b）原触珠蛋白和核心糖基化β亚基（Mr = 约35000）的碳水化合物侧链在高尔基体中转化为复杂的、唾液酸化的侧链，导致形成完全糖基化的原触珠蛋白（Mr = 约48000）和β亚基（Mr = 约38000），并且这些蛋白形式以及α亚基（Mr = 约9500）被分泌；（c）用衣霉素抑制糖基化对分离的肝细胞中各种触珠蛋白多肽的合成、加工或分泌速率没有显著影响；（d）在秋水仙碱存在下进行的类似实验对中间体的合成和加工速率也没有影响；（e）体内和从分离的肝细胞分泌的触珠蛋白种类约60 - 70%为α2β2四聚体形式，其余为二聚化的原触珠蛋白。据推测，分泌的原触珠蛋白可能在分泌后被加工成天然亚基结构，因为我们证明，将原触珠蛋白与正常大鼠血清、大鼠血浆或其他动物物种的血清一起孵育会导致其转化为天然蛋白的相应α和β亚基。
