title: Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line.
abstract: Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan. Electronic address: s47526a@cc.nagoya-u.ac.jp.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Hirako
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yonemoto
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Tomoe
authors.initials: T
authors.lastname: Yamauchi
authors.affiliation: Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nishizawa
authors.affiliation: Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kawamoto
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Katsushi
authors.initials: K
authors.lastname: Owaribe
mesh_terms: D001324:Autoantigens / Q000302:isolation & purification / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000648:ultrastructure*; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules / Q000302:isolation & purification / Q000378:metabolism; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D022002:Hemidesmosomes / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D039503:Integrin alpha6 / Q000302:isolation & purification / Q000378:metabolism; D039663:Integrin beta4 / Q000302:isolation & purification / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens / Q000302:isolation & purification / Q000378:metabolism; D051190:Plectin / Q000302:isolation & purification / Q000378:metabolism; D012878:Skin Neoplasms / Q000648:ultrastructure*; D013347:Subcellular Fractions; D000097794:Kalinin; D000097805:Collagen Type XVII
chemical_list: D001324:Autoantigens; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D039503:Integrin alpha6; D039663:Integrin beta4; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D051190:Plectin; D000097794:Kalinin; D000097805:Collagen Type XVII; C486268:DST protein, human
keywords: Autoimmune disease; Basement membrane; Hemidesmosome; Keratinocyte; Laminin