Post-translational modifications and binding properties of the apically secreted 80-kDa glycoprotein from Madin-Darby canine kidney cells: similarities to the C-terminal portion of the basolaterally secreted fibronectin.
Apically secreted 80-kDa glycoprotein (gp 80) from Madin-Darby canine kidney cells was found to be immunoprecipitated by the polyclonal antiserum against fibronectin or a monoclonal antibody specific for the fibronectin C-terminal fibrin binding domain. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gp 80 migrated as a doublet band under nonreducing conditions. Under reducing conditions, gp 80 was resolved into three distinct bands, respectively of 45-, 40-, and 35-kDa molecular mass. Analysis by two-dimensional SDS-PAGE revealed that gp 80 exists in two molecular forms: one consisting of a 45-kDa subunit and a 40-kDa subunit, and one consisting of a 45-kDa subunit and a 35-kDa subunit. V-8 protease mapping indicated the 40 and 35-kDa subunits as being of the same homologous group and also as bearing partial homology to the 45-kDa subunit. Radioactive labeling revealed that labeled gp 80 was subjected to covalent modifications by sulfation and phosphorylation. Sulfate analysis showed that [35S]sulfate-labeled gp 80 contained ca. 2.45 +/- 0.07% tyrosine-bound [35S]sulfate with the rest being presumably carbohydrate-bound. [32P]-Phosphate-labeled gp 80, on the other hand, was found to contain serine-O-phosphate as the predominant phosphorylated amino acid residue. Employing the affinity gel fractionation technique, it was shown that gp 80 exhibited binding affinities toward heparin and fibrin. Binding of gp 80 to heparin-agarose or fibrin-Sepharose, however, was inhibited in the presence of added fibronectin or the monoclonal antibody. Tryptic peptide mapping revealed common peptide spots between fibronectin and the three subunits of gp 80. Furthermore, Western blot analysis showed that fibronectin could be recognized and bound by anti-gp 80 antibodies. These results indicate that gp 80 bears both structural and functional similarities to the C-terminal portion of the fibronectin molecule.
Han
J R
JR
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Suiko
M
M
Liu
C C
CC
Liu
M C
MC
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004285:Dogs; D005353:Fibronectins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007668:Kidney; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D013431:Sulfates / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D005353:Fibronectins; D006023:Glycoproteins; D013431:Sulfates; C005662:tyrosine O-sulfate; D014443:Tyrosine