Synthesis and glycosylation of fibronectin in hepatocytes from vitamin A-deficient rats

Summary Recent work from our laboratory (Kim and Wolf, J Biol Chem 262: 365–371, 1987) has shown increased uptake of labeled amino acids into fibronectin (FN), increased net synthesis of FN and increased levels of FN-mRNA in primary cultures of hepatocytes from vitamin A-deficient rats compared to controls. We now find, surprisingly, decreased uptake of labeled sugars into the oligosaccharide chains of FN from vitamin A-deficient hepatocytes. This decrease could be reversed by added retinoic acid at physiological concentration. At the same time, FN from deficient hepatocytes (–A.FN) was more susceptible to proteolytic degradation. Decreased uptake of the core sugar mannose into –A.FN was similar to that of glucosamine, yet the percent of label in sialic acid was the same as in +A.FN, suggesting a smaller number of oligosaccharide chains per molecule of –A.FN. Upon enzymatic removal of oligosaccharide and labeling with sodium borotritide, it was found that both –A.FN and +A.FN had biantennary oligosaccharide structures. Selective enzymatic removal of sialic acid showed that +A.FN had both sialic acids in an 23 linkage, whereas –A.FN apparently had one 23 and one 26-linked sialic acid. The borotritide experiments allowed us to calculate that +A.FN appeared to have 5 oligosaccharide chains per FN monomer, whereas the –A. FN showed only 4 chains. These results would account for the decreased glycosylation and increased susceptibility to proteolysis of the –A. FN. We conclude that vitamin A controls both the rate of synthesis of the polypeptide chain of FN via its mRNA, as well as the rate of its glycosylation.Abbreviations FN Fibronectin - ELISA Enzyme-linked Immunosorbent Assay - DOC Deoxycholate - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride - PBS Phosphate-buffered Saline - BSA Bovine Serum Albumin - AGP Alpha-1 acid Glycoprotein - SDS-PAGE Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis