Decastel M, Doyennette-Moyne M A, Gouet E, Aubery M, Codogno P
CNRS UAC 71, INSERM U180, UFR Biomédicale des Saints-Pères, Paris, France.
Biochem J. 1993 Apr 1;291 ( Pt 1)(Pt 1):247-55. doi: 10.1042/bj2910247.
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.
扎伊德氏肝癌细胞是源自未分化肿瘤并移植到大鼠体内的贴壁性差的细胞。我们比较了正常大鼠肝细胞和扎伊德氏肝癌细胞中纤连蛋白受体的生物合成、结构和功能。大鼠肝细胞纤连蛋白受体已被分离出来。它由两个亚基组成：α5（分子量155 kDa）和β1（分子量115 kDa）。然而，其生物合成尚未见报道。使用针对受体各亚基产生的多克隆抗体，我们观察到α5亚基在正常大鼠肝细胞和扎伊德氏肝癌细胞中均作为155 kDa的多肽合成。相比之下，扎伊德氏肝癌细胞中β1亚基的分子量为130 kDa，而正常大鼠肝细胞中为115 kDa。脉冲追踪实验表明，扎伊德氏肝癌细胞中从100 kDa的β1前体到130 kDa成熟形式的明显转变时间异常延长，因为直到24小时后才检测到后者，而正常大鼠肝细胞中从100 kDa前体到115 kDa成熟形式的转变在3小时内就开始了。用内切β-N-乙酰葡糖胺糖苷酶H和肽N-糖苷酶消化正常大鼠肝细胞和扎伊德氏肝癌细胞的100 kDaβ1前体，通过SDS/PAGE判断，分别产生了从100 kDa到84 kDa和82 kDa的产物，这表明正常大鼠肝细胞和扎伊德氏肝癌细胞中合成的是同一条多肽链。用肽N-糖苷酶孵育成熟的正常大鼠肝细胞β1亚基，通过SDS/PAGE判断，其分子量从115 kDa降至82 kDa，而异常成熟的扎伊德氏肝癌细胞β1亚基的分子量从130 kDa降至110 kDa。因此，除了天冬酰胺连接的寡糖加工改变外，“腹水生长”还在扎伊德氏肝癌细胞β1亚基中诱导了其他翻译后修饰。此外，在扎伊德氏肝癌细胞表面检测到了异常成熟的130 kDa和前体100 kDa的β1亚基，它们与α5亚基相关联。本文讨论了纤连蛋白受体的这些结构改变与本研究中观察到的扎伊德氏肝癌细胞与可溶性纤连蛋白或包被的纤连蛋白基质结合受损之间的关系。
