Studies on the Biosynthesis of Laminin by Murine Parietal Endoderm Cells

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). Copyright © 1981, Wiley Blackwell. All rights reserved