Stable expression of human tartrate-resistant acid phosphatase isoforms by CHO cells.
OBJECTIVE
In human serum, type-5 tartrate-resistant acid phosphatase (TRACP) exists as two closely related isoforms: 5a and 5b. Serum isoform 5b is an osteoclast product that reflects bone resorption rate and is frequently increased in diseases of increased bone turnover. Isoform 5a protein is often increased in rheumatoid arthritis (RA) sera and may be a product of inflammatory macrophages. Our objective was to compare the biochemical characteristics of TRACP 5a and 5b.


METHODS
We transfected the human ACP 5 gene into CHO cells and cloned a stable cell line (CHO/TRACP 8F5) that expresses high levels of TRACP activity both intracellularly and as a secreted product. Both enzyme preparations were purified on an anti-TRACP antibody column. Their biochemical properties were compared to the natural serum isoforms using colorimetric assays for activity and total protein. Their structural properties were compared to natural serum isoforms using denaturing and nondenaturing polyacrylamide gel electrophoresis.


RESULTS
Both enzyme preparations were heterogeneous. The combined secreted recombinant TRACPs (rTRACP(ex)) had all the characteristics of natural serum TRACP 5a. There were seven uncleaved glycoproteins with a pH optimum of 5.2, relatively low specific activity (278 U/mg) and differentially sialylated. The combined intracellular TRACPs (rTRACP(in)) had all the characteristics of natural serum TRACP 5b. They consisted of two proteins, one of which was a processed heterodimer, with a pH optimum of 5.8, a relatively high specific activity (887 U/mg) and lacked sialic acid.


CONCLUSIONS
This cell line provides an avenue for the simultaneous study of the regulation, function and intracellular trafficking of separate TRACP isoforms and the identification of their physiologic substrates in a single uniform cell source.
Janckila
Anthony J
AJ
Special Hematology Laboratory, Veterans Administration Medical Center, 800 Zorn Avenue (151), Louisville, KY 40206, USA. anthony.janckila@med.va.gov
Parthasarathy
Ranga N
RN
Parthasarathy
Latha K
LK
Seelan
Ratnam S
RS
Yam
Lung T
LT
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006651:Histocytochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; C000605510:ACP5 protein, human; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase; D009597:4-Nitrophenylphosphatase