Mutational analysis of the carbohydrate binding activity of the tobacco lectin

Abstract

At present the three-dimensional structure of the tobacco lectin, further referred to as Nictaba, and its carbohydrate-binding site are unresolved. In this paper, we propose a three-dimensional model for the Nictaba domain based on the homology between Nictaba and the carbohydrate-binding module 22 of Clostridium thermocellum Xyn10B. The suggested model nicely fits with results from circular dichroism experiments, indicating that Nictaba consists mainly of β-sheet. In addition, the previously identified nuclear localization signal is located at the top of the protein as a part of a protruding loop. Judging from this model and sequence alignments with closely related proteins, conserved glutamic acid and tryptophan residues in the Nictaba sequence were selected for mutational analysis. The mutant DNA sequences as well as the original Nictaba sequence have been expressed in Pichia pastoris and the recombinant proteins were purified from the culture medium. Subsequently, the recombinant proteins were characterized and their carbohydrate binding properties analyzed with glycan array technology. It was shown that mutation of glutamic acid residues in the C-terminal half of the protein did not alter the carbohydrate-binding activity of the lectin. In contrast, mutation of tryptophan residues in the N-terminal half of the Nictaba domain resulted in a complete loss of carbohydrate binding activity. These results suggest that tryptophan residues play an important role in the carbohydrate binding site of Nictaba.

Evolution and structural diversification ofNictaba-like lectin genes in food crops with a focus on soybean (Glycine max)

“…Structurally related proteins often share similar molecular functions (Brylinski and Skolnick, 2008;Drew et al, 2011;Rentzsch and Orengo, 2013). This is supported by two tryptophan resisdues, which were shown to be indispensable for lectin activity of Nictaba, are conserved in most of the GmNictaba domain sequences (Schouppe et al, 2010). These Nictaba homologues most likely represent functional carbohydrate-binding proteins.…”

“…Analysis revealed that, like Nictaba (Schouppe et al, 2010), the soybean homologue also consists of b-sheets. Structurally related proteins often share similar molecular functions (Brylinski and Skolnick, 2008;Drew et al, 2011;Rentzsch and Orengo, 2013).…”

“…Molecular modelling of an NLL protein from soybean reveals structural resemblance to the tobacco lectin Despite the lack of a three-dimensional structure of the tobacco lectin, the availability of a structure model of Nictaba accommodated new insights in some amino acid residues that are important for the carbohydrate-binding activity of the lectin (Schouppe et al, 2010). Molecular models built for Nictaba and a selected soybean Nictaba homologue (GmNLL1) Glyma.…”

“…7. Interestingly, the two tryptophan residues that are necessary for the carbohydrate-binding activity of the tobacco lectin (Schouppe et al, 2010) are strongly conserved in the soybean NLL sequences (Fig. 7, positions 17 and 28).…”

Interaction of the Tobacco Lectin with Histone Proteins

“…In the future, the interaction of Nictaba with histone proteins will have to be confirmed by other biochemical, genetic, and microscop- ical techniques. Negative control experiments with a Nictaba mutant protein affected in its carbohydratebinding activity (Schouppe et al, 2010) could also be useful to prove the interaction with histone proteins.…”