Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase

• Published in: PloS one Vol. 11; no. 11; p. e0165227
• Main Authors: Shapiro, Jason M, Chung, Waihong, Ogawa, Kosuke, Barker, Luke, Carlson, Rolf, Wands, Jack R, Li, Jisu
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-11-2016; Public Library of Science (PLoS)
• Subjects: Adenosine Triphosphatases - metabolism; Analysis; Animals; Antigens; Antigens, Neoplasm - metabolism; ATPases; Bioindicators; Biological Transport - physiology; Biology and Life Sciences; Biomarkers; Biotechnology; Cancer; Cancer therapies; Carcinoma, Hepatocellular - metabolism; Cell Line; Cell Line, Tumor; Cell surface; Cellulose; Cercopithecus aethiops; Chromatography; Colon; Colon cancer; Colonic Neoplasms - metabolism; Colorectal cancer; Complex formation; COS Cells; Drug delivery; Drug delivery systems; Drugs; Gel electrophoresis; Glycoproteins; Glycosylation; Health aspects; Heat exchange; Heat shock proteins; Hep G2 Cells; Hepatoma; HSP90 Heat-Shock Proteins - metabolism; Hsp90 protein; Humans; Immunoglobulins; Immunoprecipitation; Ion-exchange chromatography; Iron; Iron - metabolism; Leukemia; Liver cancer; Liver Neoplasms - metabolism; Localization; Mass spectrometry; Mass spectroscopy; Medical schools; Medicine and Health Sciences; Mice; Molecular structure; Monoclonal antibodies; Na+/K+-exchanging ATPase; NIH 3T3 Cells; Protein Binding - physiology; Protein transport; Proteins; Receptors, Transferrin - metabolism; Research and Analysis Methods; Rodents; Thyroid gland; Tissues; Transfection; Transferrin; Transferrin - metabolism; Transferrins; Trypsin; Tumor cell lines; Tumors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0165227

We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.