Biosynthesis, surface expression and function of the fibronectin receptor after rat liver cell transformation to tumorigenicity

Abstract

Zajdela hepatoma cells are poorly-adherent cells derived from an undifferentiated tumour and transplanted into rat. We compared the biosynthesis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte fibronectin receptor has been isolated. It is composed of two subunits: alpha 5 (molecular mass 155 kDa) and beta 1 (molecular mass 115 kDa). However, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we observed that the alpha 5-subunit was synthesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta 1-subunit was 130 kDa in Zajdela hepatoma cells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta 1-precursor to the 130-kDa mature form was abnormally prolonged in Zajdela hepatoma cells since the latter was not detected until 24 h, while the transition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta 1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized in normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta 1-subunit with peptide N-glycosidase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SDS/PAGE, while the molecular mass of the abnormal mature Zajdela hepatoma cell beta 1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, ‘ascitic growth’ induced other post-translational modifications in the Zajdela hepatoma cell beta 1-subunit. Furthermore, both the abnormal mature 130-kDa and precursor 100-kDa beta 1-subunits were detected on the surface of Zajdela hepatoma cells, associated with the alpha 5-subunit. The relationship between these structural alterations in the fibronectin receptor and the impaired Zajdela hepatoma cell binding to soluble fibronectin or to a coated fibronectin matrix that was observed in this study is discussed.