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