Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes

Abstract

One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment ( E. M. Sjöberg and E. Fries, 1990, Biochem. J. 272, 113–118 ). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-α-inhibitor and the inter-α-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to α 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [ 35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-α-inhibitor), and one >240 kDa (possibly a protein related to the inter-α-inhibitor). Pulse-chase experiments with [ 35S]sulfate showed that these proteins occurred intracellularly as precursors containing α 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the α 1-microglobulin/bikunin precursor.