Biosynthesis and processing of rat haptoglobin.

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.