Mutational analysis of the carbohydrate binding activity of the tobacco lectin.
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.
Schouppe
Dieter
D
Department of Molecular Biotechnology, Laboratory of Biochemistry and Glycobiology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
Rougé
Pierre
P
Lasanajak
Yi
Y
Barre
Annick
A
Smith
David F
DF
Proost
Paul
P
Van Damme
Els J M
EJ
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism / Q000235:genetics*; D002240:Carbohydrate Sequence; D017124:Conserved Sequence / Q000235:genetics; D004252:DNA Mutational Analysis; D018698:Glutamic Acid / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D037102:Lectins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014364:Tryptophan