title: Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans.
abstract: The possible in vivo interaction of the Nicotiana tabacum agglutinin (Nictaba) with endogenous glycoproteins was corroborated using a combination of confocal/electron microscopy of an EGFP-Nictaba fusion protein expressed in tobacco Bright Yellow-2 (BY-2) cells and biochemical analyses. In vitro binding studies demonstrated that the expressed EGFP-Nictaba possesses carbohydrate-binding activity. Microscopic analyses confirmed the previously reported cytoplasmic/nuclear location of Nictaba in jasmonate-treated tobacco leaves and provided evidence for the involvement of a nuclear localization signal-dependent transport mechanism. In addition, it became evident that the lectin is not uniformly distributed over the nucleus and the cytoplasm of BY-2 cells. Far Western blot analysis of extracts from whole BY-2 cells and purified nuclei revealed that Nictaba interacts in a glycan inhibitable way with numerous proteins including many nuclear proteins. Enzymatic deglycosylation with PNGase F indicated that the observed interaction depends on the presence of N-glycans. Glycan array screening, which showed that Nictaba exhibits a strong affinity for high-mannose and complex N-glycans, provided a reasonable explanation for this observation. The cytoplasmic/nuclear localization of a plant lectin that has a high affinity for high-mannose and complex N-glycans and specifically interacts with conspecific glycoproteins suggests that N-glycosylated proteins might be more important in the cytoplasm and nucleus than is currently believed.
authors.affiliation: Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
authors.forename: Nausicaä
authors.initials: N
authors.lastname: Lannoo
authors.forename: Willy J
authors.initials: WJ
authors.lastname: Peumans
authors.forename: Els Van
authors.initials: EV
authors.lastname: Pamel
authors.forename: Rick
authors.initials: R
authors.lastname: Alvarez
authors.forename: Tou-Cheu
authors.initials: TC
authors.lastname: Xiong
authors.forename: Gerd
authors.initials: G
authors.lastname: Hause
authors.forename: Christian
authors.initials: C
authors.lastname: Mazars
authors.forename: Els J M
authors.initials: EJ
authors.lastname: Van Damme
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D008358:Mannose / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D037121:Plant Lectins / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D037121:Plant Lectins; D011134:Polysaccharides; D008358:Mannose