title: Monoclonal antibodies recognize gly-leu-phe-gly repeat of nucleoporin nup98 of tetrahymena, yeasts, and humans.
abstract: Nucleoporin Nup98, an essential component of the nuclear pore complex, has multifunctional roles in nuclear functions including transcriptional regulation and nucleocytoplasmic transport. These functions mostly depend on a Gly-Leu-Phe-Gly (GLFG) sequence appearing repetitively in the N-terminal region of Nup98. As the GLFG sequence is well conserved among Nup98s from a wide variety of species including humans, yeasts, and ciliates such as Tetrahymena thermophila, a specific antibody that recognizes the GLFG sequence is expected to detect various Nup98s from a wide-range of species. To generate monoclonal antibodies specific to the GLFG repeat of Nup98, we used two synthetic polypeptides derived from the macronuclear Nup98 of T. thermophila as an antigen. We obtained two monoclonal antibodies (MAbs), 13C2 and 21A10, that recognize Nup98s in indirect immunofluorescence staining and Western blot analysis of T. thermophila. Peptide array analysis of these monoclonal antibodies located the position of their epitopes at or near GLFG residues: the epitope recognized by the 13C2 MAb is FGxxN (x being any amino acid), and the epitope recognized by the 21A10 MAb is GLF. As expected by their epitopes, these monoclonal antibodies also recognize Nup98 homologs expressed by human cells and the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, indicating that 13C2 and 21A10 MAbs recognize Nup98 epitopes common to phylogenetically distinct organisms. Thus, these MAbs are useful in studying a wide variety of biological phenomena that involve Nup98, ranging from ciliate nuclear dimorphism to NUP98-related human leukemia.
authors.affiliation: Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology (NICT), Kobe, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwamoto
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Asakawa
authors.forename: Chizuru
authors.initials: C
authors.lastname: Ohtsuki
authors.forename: Hiroko
authors.initials: H
authors.lastname: Osakada
authors.forename: Takako
authors.initials: T
authors.lastname: Koujin
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D000918:Antibody Specificity; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry / Q000276:immunology*; D015800:Protozoan Proteins / Q000276:immunology*; D020449:Repetitive Sequences, Amino Acid / Q000276:immunology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000276:immunology*; D012568:Schizosaccharomyces; D029702:Schizosaccharomyces pombe Proteins / Q000276:immunology*; D016808:Tetrahymena thermophila
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D028861:Nuclear Pore Complex Proteins; C465451:Nup98 protein, human; D015800:Protozoan Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins