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Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high‐mannose and complex N ‐glycans
Author(s) -
Lannoo Nausicaä,
Peumans Willy J.,
Pamel Els Van,
Alvarez Rick,
Xiong Tou-Cheu,
Hause Gerd,
Mazars Christian,
Van Damme Els J.M.
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.10.044
Subject(s) - cytoplasm , glycan , nicotiana tabacum , lectin , biochemistry , biology , glycoprotein , mannose , microbiology and biotechnology , fusion protein , nuclear transport , cell nucleus , recombinant dna , gene
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.