Studies on the Biosynthesis of Laminin by Murine Parietal Endoderm Cells

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Abstract
The biosyntnesis and processing of the polypeptides A (Mr=450×103), B1 (Mr=450×103), B2 (Mr=230×103) and C (Mr=150×103) of the extracellular matrix protein, laminin, were studied in murine parietal endoderm cells labeled with [35S]methionine. Various lines of evidence suggest that the A chains are not precursors to the smaller B chains. Firstly, in pulse-chase experiments, radioactivity in cytoplasmic A and (B1+ B2) chains declines with the same half-life of about 70 min. Secondly, peptide maps generated by digestion of A and (B1+ B2) chains with Staphylococcus aureus V8 protease are different. Finally, rabbit antibodies to isolated, denatured (B1+ B2) chains do not cross-react with reduced and alkylated A chains. A, B1, B2 and C polypeptides are all glycosylated by an intracellular process involving the addition of tunicamycin and endo β-N-acetylglucosaminidase H. sensitive N-linked oligosaccharide side chains. Further glycosylation probably occurs around the time of secretion. Disulphide bonding of some A and B chains can be observed in the cytoplasm within 10 min of adding [35S]methionine. However, it appears that some free A and B2 chains are present in the cytoplasm and that free A chains exist in the medium. The relationship between the 150×103-Mr C glycoprotein and the A and B components is discussed. Although B and C chains generate different peptide maps after digestion with S, aureus V8 protease, antibodies raised against isolated, denatured C chains cross-react with reduced and alkylated B (but not A) chains. This suggests that B and C chains may share some antigenic determinant(s).