This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, J. Articles by Rapoport, B. Search for Related Content PubMed PubMed Citation Articles by Guo, J. Articles by Rapoport, B.

The Greater Glycan Content of Recombinant Human Thyroid Peroxidase of Mammalian Than of Insect Cell Origin Facilitates Purification to Homogeneity of Enzymatically Protein Remaining Soluble at High Concentration¹

Thyroid Molecular Biology Unit, Veterans Administration Medical Center and University of California (J.G., S.M.M., B.R.), San Francisco, California 94121; and Nichols Institute Diagnostics (S.H.), San Juan Capistrano, California 92690

Address all correspondence and requests for reprints to: Dr. Basil Rapoport, M.D., Thyroid Molecular Biology Unit (111T), VeteransAdministration Medical Center, 4150 Clement Street, San Francisco, California 94121.

Structural studies on thyroid peroxidase (TPO), a major thyroid autoantigen, require milligram amounts of pure protein. We found that the human TPO ectodomain (amino acid residues 1–848) generated in insect cells did not remain in solution at high concentrations after affinity purification. In contrast, the TPO ectodomain secreted by mammalian (Chinese hamster ovary) cells, although generated to a lesser extent (1 vs. 8 mg/liter), remained in solution at high concentration (10 mg/ml) after purification to homogeneity. This purified material was well recognized by TPO autoantibodies, but lacked enzymatic activity. We attempted to restore activity by culturing the Chinese hamster ovary cells in the presence of added heme. TPO enzymatic activity was clearly detected in conditioned medium from cells cultured in hematin and hemin, but not in protoporphyrin IX (all at 1 mg/liter). Heme prosthetic group incorporation into affinity-purified TPO was highest for hematin and hemin, but unchanged for protoporphyrin IX (OD 410/280 nm ratios of 0.25, 0.23, and 0.14, respectively). Enzymatic activity was now evident with hemin (mean ± SE, 27.2 ± 2.6; n = 3; guaiacol units/mg protein), hematin (24.1 ± 1.6), and, to a lesser extent, protoporphyrin IX (3.6 ± 0.2). Culturing cells in 20 mg/liter hematin, the maximum concentration tolerated, increased enzymatic activity even further (45.6 ± 0.6 guaiacol units/mg protein). All purified TPO preparations were homogeneous on PAGE and of similar size (105 kDa). Enzymatic deglycosylation showed a complex carbohydrate contribution of 13 kDa (unlike the 2.3 kDa in insect cell TPO).

In conclusion, this is the first report on the purification to homogeneity of recombinant human TPO of mammalian cell origin. Unlike TPO generated in insect cells, mammalian TPO remains soluble at high concentration, possibly because of its greater carbohydrate content. This enzymatically active, recombinant human TPO may be useful for future structural studies.

This article has been cited by other articles:

				P. N. Pichurin, C.-R. Chen, G. D. Chazenbalk, H. Aliesky, N. Pham, B. Rapoport, and S. M. McLachlan Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization J. Immunol., January 1, 2006; 176(1): 668 - 676. [Abstract] [Full Text] [PDF]

				J. Guo, S. M. McLachlan, P. N. Pichurin, C.-R. Chen, N. Pham, H. A. Aliesky, C. S. David, and B. Rapoport Relationship between Thyroid Peroxidase T Cell Epitope Restriction and Antibody Recognition of the Autoantibody Immunodominant Region in Human Leukocyte Antigen DR3 Transgenic Mice Endocrinology, November 1, 2005; 146(11): 4961 - 4967. [Abstract] [Full Text] [PDF]

				J. Guo, P. N. Pichurin, J. C. Morris, B. Rapoport, and S. M. McLachlan "Naked" Deoxyribonucleic Acid Vaccination Induces Recognition of Diverse Thyroid Peroxidase T Cell Epitopes Endocrinology, August 1, 2004; 145(8): 3671 - 3678. [Abstract] [Full Text] [PDF]

				F. Latrofa, P. Pichurin, J. Guo, B. Rapoport, and S. M. McLachlan Thyroglobulin-Thyroperoxidase Autoantibodies Are Polyreactive, Not Bispecific: Analysis Using Human Monoclonal Autoantibodies J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 371 - 378. [Abstract] [Full Text] [PDF]

				U. Haberkorn and A. Altmann Imaging Techniques for Gene Therapy: SPECT, PET, and MRI Journal of Pharmacy Practice, October 1, 2001; 14(5): 383 - 396. [Abstract] [PDF]

				J. Guo, X.-M. Yan, S. M. McLachlan, and B. Rapoport Search for the Autoantibody Immunodominant Region on Thyroid Peroxidase: Epitopic Footprinting with a Human Monoclonal Autoantibody Locates a Facet on the Native Antigen Containing a Highly Conformational Epitope J. Immunol., January 15, 2001; 166(2): 1327 - 1333. [Abstract] [Full Text] [PDF]

				J. C. Jaume, J. Guo, Y. Wang, B. Rapoport, and S. M. McLachlan Cellular Thyroid Peroxidase (TPO), Unlike Purified TPO and Adjuvant, Induces Antibodies in Mice That Resemble Autoantibodies in Human Autoimmune Thyroid Disease J. Clin. Endocrinol. Metab., May 1, 1999; 84(5): 1651 - 1657. [Abstract] [Full Text]

				L. Fayadat, P. Niccoli-Sire, J. Lanet, and J.-L. Franc Role of Heme in Intracellular Trafficking of Thyroperoxidase and Involvement of H2O2 Generated at the Apical Surface of Thyroid Cells in Autocatalytic Covalent Heme Binding J. Biol. Chem., April 9, 1999; 274(15): 10533 - 10538. [Abstract] [Full Text] [PDF]

				X. Zhang and P. Arvan Cell Type-dependent Differences in Thyroid Peroxidase Cell Surface Expression J. Biol. Chem., October 6, 2000; 275(41): 31946 - 31953. [Abstract] [Full Text] [PDF]