Rabbit Anti-Human ACP5 Polyclonal Antibody

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. (c) 2007 Elsevier Inc. All rights reserved.

Tartrate-resistant acid phosphatase, although encoded by a single gene, exists as two isoforms in human serum, TRAP 5a and 5b, differing in post-translational modifications such as proteolytic processing and kinetic properties including pH optimum and specific activity. The biogenetic relationship between the TRAP isoforms was assessed in a stably transfected breast cancer epithelial MDA-MB-231 cell subline overexpressing 5a- and 5b-like TRAP isoforms intracellularly, with only the monomeric 5a-like isoform being secreted. As judged by immunolocalization and comparative N-glycan profiling by Con A lectin chromatography and glycanase analysis, the majority of the intracellular monomeric TRAP was destined for secretion, while a minor portion provided the putative precursor for the intracellular 5b-like isoform. Brefeldin A blocked secretion of 5a-like TRAP isoform as well as appearance of its putative intracellular precursor, and augmented the intracellular level of proteolytically processed 5b-like isoform, indicating a common early biosynthetic precursor for TRAP isoforms 5a and 5b. The cysteine proteinase inhibitor E64 partially blocked formation of the 5b-like isoform while augmenting the level of its putative monomeric precursor, but did not alter the levels of secreted TRAP or its intracellular precursor, suggesting that distinct precursors for secreted TRAP 5a and intracellular 5b-like isoform are segregated in the ER or Golgi prior to proteolytic processing. In conclusion, these data provide evidence that distinct monomeric TRAP populations are diverted early in the secretory pathway either giving rise to a secreted, monomeric 5a-like TRAP isoform or to an intracellular, proteolytically processed 5b-like TRAP isoform. (C) 2010 Elsevier B.V. All rights reserved.