Expression and proteolytic processing of mammalian purple acid phosphatase in CHO-K1 cells.
Rat recombinant purple acid phosphatase (PAP) stably expressed in fibroblast-like CHO-K1 cells was purified and characterized with respect to post-translational modifications such as N-glycosylation and proteolytic processing in order to elucidate subcellular and molecular pathways for proteolytic activation. In these cells, proteolytically processed PAP was more abundant than the monomeric form. PAP-transfected CHO-K1 cells were expressing active cathepsin K intracellularly, which was partially co-localized with PAP. However, neither cathepsin K nor trypsin digestion of the purified monomeric PAP in vitro did result in a two-subunit form with kinetic and electrophoretic properties resembling the endogenous cellular two-subunit form. Treatment of PAP-transfected CHO-K1 cells with the cysteine proteinase inhibitor E-64 suggested that only a minor fraction of secreted PAP is processed intracellularly by cysteine proteinases. These data do not support a dominant or critical role for cathepsins or trypsin-like serine proteinases in the proteolytic activation of PAP in CHO-K1 cells, implicating yet unidentified proteinases in the proteolytic processing of both intracellular and secreted PAP in this cell line.
Wang
Yunling
Y
Karolinska Institutet, Department of Laboratory Medicine, Division of Pathology, Karolinska University Hospital, S-141 86 Huddinge, Sweden.
Andersson
Göran
G
D000135:Acid Phosphatase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004795:Enzyme Stability / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats
D006023:Glycoproteins; D007527:Isoenzymes; C091137:purple acid phosphatase; D000135:Acid Phosphatase; D010447:Peptide Hydrolases