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.

Interaction of the Tobacco Lectin with Histone Proteins

“…These results are in agreement with previous observations that showed the specific interaction of Nictaba with GlcNAc oligomers (Lannoo et al, 2006b). Future studies will concentrate on the ultrastructural colocalization of Nictaba and histone proteins in the plant cell and the functional implications resulting from this interaction.…”

“…The extracellular compartment of plant cells contains many heavily glycosylated proteins (Knox, 1995;Jose-Estanyol and Puigdomenech, 2000;Léonard et al, 2002). Taking into account the carbohydratebinding properties of Nictaba as determined by glycan array analyses (Lannoo et al, 2006b), it is not surprising that these proteins are retained on the lectin column. Therefore, a more rigid purification and fractionation of tobacco cells was performed.…”

“…Using immunohistochemistry and confocal microscopy with a GFP-Nictaba fusion protein, it was confirmed that Nictaba resides in the nucleocytoplasmic compartment of the plant cell and is virtually absent from the nucleolus (Chen et al, 2002;Lannoo et al, 2006b;Vandenborre et al, 2009). Using hapten inhibition assays and glycan array technology, it was demonstrated that Nictaba exhibits specificity for GlcNAc oligomers but also interacts with high-Man and complex N-glycans (Lannoo et al, 2006b).…”

“…Immunodetection was achieved by a colorimetric assay using 3,3#-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) as a substrate (Lannoo et al, 2006b). Inhibition of Nictaba binding was achieved by a 1-h preincubation of Nictaba with a mixture of GlcNAc oligomers (at a final concentration 10-fold higher than that required to completely inhibit the agglutination activity of a 100 mg mL 21 solution of Nictaba; Lannoo et al, 2006b).…”

Functional Characterization of HFR1, a High-MannoseN-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae

“…If high-Man glycoproteins are also present in Hessian fly larval salivary glands or gustatory receptors, then it is conceivable that high concentrations of His 6 -HFR1 may act as an antifeedant by binding to these structures; thus, they would not be ingested. His 6 -HFR1 does not require the core di-GlcNAc of the N-glycans for binding (Supplemental Table S1), as required by some other high-Man N-glycan-binding lectins such as tomato (Solanum lycopersicum) lectin (Lycopersicon esculentum agglutinin; Oguri, 2005) and tobacco (Nicotiana tabacum) lectin (Nicotiana tabacum agglutinin; Chen et al, 2002;Lannoo et al, 2006). The positive hemagglutination activity suggested that the presence of the His tag did not interfere with His 6 -HFR1 function (Supplemental Fig.…”