title: Nanog1 in NTERA-2 and recombinant NanogP8 from somatic cancer cells adopt multiple protein conformations and migrate at multiple M.W species.
abstract: Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Bigang
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Badeaux
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Grace
authors.initials: G
authors.lastname: Choy
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Dhyan
authors.initials: D
authors.lastname: Chandra
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Irvin
authors.initials: I
authors.lastname: Shen
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Collene R
authors.initials: CR
authors.lastname: Jeter
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Kiera
authors.initials: K
authors.lastname: Rycaj
authors.affiliation: College of Pharmacy, University of Texas, Austin, Texas, United States of America.
authors.forename: Chia-Fang
authors.initials: CF
authors.lastname: Lee
authors.affiliation: College of Pharmacy, University of Texas, Austin, Texas, United States of America.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Person
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Can
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Yueping
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Shen
authors.affiliation: Department of Biochemistry, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Sung Yun
authors.initials: SY
authors.lastname: Jung
authors.affiliation: Department of Biochemistry, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.affiliation: Department of Molecular Carcinogenesis, University of Texas M.D Anderson Cancer Center, Science Park, Smithville, Texas, United States of America; Cancer Stem Cell Institute, Research Center for Translational Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China.
authors.forename: Dean G
authors.initials: DG
authors.lastname: Tang
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D055785:Gene Knockdown Techniques; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000071317:Nanog Homeobox Protein; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D020033:Protein Isoforms