Biosynthesis of the adenosine deaminase-binding protein in human fibroblasts and hepatoma cells.
The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis*; D002460:Cell Line; D018819:Dipeptidyl Peptidase 4; D005347:Fibroblasts / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007163:Immunosorbent Techniques; D007668:Kidney / Q000378:metabolism; D007680:Kidney Neoplasms / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism*; D008253:Macaca mulatta; D009844:Oligosaccharides / Q000032:analysis
D002352:Carrier Proteins; D009844:Oligosaccharides; D018819:Dipeptidyl Peptidase 4