Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high-mannose and complex N-glycans.
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.
Lannoo
Nausicaä
N
Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
Peumans
Willy J
WJ
Pamel
Els Van
EV
Alvarez
Rick
R
Xiong
Tou-Cheu
TC
Hause
Gerd
G
Mazars
Christian
C
Van Damme
Els J M
EJ
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
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D037121:Plant Lectins; D011134:Polysaccharides; D008358:Mannose