Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line.
We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.
Ullrich
O
O
Abteilung Molekulare Genetik, Universität Frankfurt, Republic of Germany.
Mann
K
K
Haase
W
W
Koch-Brandt
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism; D008969:Molecular Sequence Data; D053495:Osteopontin; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000378:metabolism
D010750:Phosphoproteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin