Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2.
We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [(14)C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP.
Katagiri
Yohko U
YU
Department of Developmental Biology, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo 157-8535, Japan. kata@nch.go.jp
Kiyokawa
Nobutaka
N
Nakamura
Kyoko
K
Takenouchi
Hisami
H
Taguchi
Tomoko
T
Okita
Hajime
H
Umezawa
Akihiro
A
Fujimoto
Junichiro
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000276:immunology; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D018236:Carcinoma, Embryonal / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D003016:Clostridium perfringens / Q000201:enzymology; D003101:Collodion / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D005690:Galactose / Q000378:metabolism; D005915:Globosides / Q000737:chemistry; D006028:Glycosphingolipids / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017474:Receptors, Laminin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055746:Stage-Specific Embryonic Antigens; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000939:Epitopes; D005915:Globosides; D006028:Glycosphingolipids; D017474:Receptors, Laminin; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D013395:Sucrose; C010163:Forssman glycolipid; D003101:Collodion; D009439:Neuraminidase; D005690:Galactose