Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

Abstract

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

Fig 1

The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C). (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.

Fig 2

Representative TPO immunostaining results obtained with a panel of monoclonal antibodies (mAbs) against human thyroid peroxidase (TPO) (A) and human serum pools (B) in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). A normal human thyroid cell line, NTHY, was used as a positive control. (A) Positive signal (red) was detected with all mAbs except the isotype control (negative control). In the cells incubated with TPOAbs obtained from autoimmune thyroid disease (AITD) patients (TPOAbs(+)), a positive signal (green) was observed but this staining was not observed when TPOAb-free serum (TPOAbs(-)) was used (negative control). Nuclei (blue) were counterstained with DAPI. Magnification: 630×. IIAb: secondary antibody; DAPI: 4′,6-diamino-2-phenylindole; NTHY: NTHY-ori 3–1 cell line.

Fig 3

The expression of lactoperoxidase (LPO) in breast tissues (A and B) and cell lines (C and D) derived from normal (184A1) and cancerous mammary tissues (MCF-7 and MDA-MB-231). (A) Representative immunohistochemical staining of human LPO in breast cancer (upper panel) and peritumoral tissues (lower panel) (magnification: 100×). (B) Human LPO expression in breast cancer and peritumoral tissues detected by immunoblotting with the 10376-1-AP antibody. Human lung and thyroid tissue (Graves’ disease case) lysates were used as a negative and positive control, respectively. For each lane, 50 μg of crude protein extract were loaded. A β-actin-specific antibody was used as a loading control. (C) LPO expression in cell lines as shown by immunofluorescent staining. Positive immunofluorescent signal (red) was detected in MDA-MB-231 and, to a lesser extent, in MCF-7 cells. No staining was detected when pre-immune rabbit IgG was used (insets). Nuclei were counterstained with DAPI (blue). Magnification: 630×. (D) LPO protein expression in cell lines analyzed by Western blot. LPO was detected in breast cancer cells, while no band was observed in normal 184A1 cells. For each lane, 10 μg of crude protein extract were loaded. A β-actin-specific antibody was used as a loading control.