Stable expression of human tartrate-resistant acid phosphatase isoforms by CHO cells.

Clin Chim Acta 2002 Dec;326(1-2):113-22

Special Hematology Laboratory, Veterans Administration Medical Center, 800 Zorn Avenue (151), Louisville, KY 40206, USA.

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.

Conclusion: 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.

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http://dx.doi.org/10.1016/s0009-8981(02)00280-2DOI Listing
December 2002
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