Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures

• Published in: FEBS open bio Vol. 2; no. 1; pp. 151 - 158
• Main Authors: Stefanowicz, Karolina, Lannoo, Nausicaä, Proost, Paul, Van Damme, Els J.M.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01-01-2012; John Wiley & Sons, Inc; Elsevier
• Subjects: Affinity chromatography; Agglutination; Amino acids; Arabidopsis; Binding sites; Carbohydrate-binding; Carbohydrates; Cell adhesion & migration; Genes; Genomes; Glycan array; Glycoproteins; Hypotheses; Jasmonic acid; Lectins; Lewis structure; Mammals; Oligosaccharides; Physiology; Plant lectin; Polysaccharides; Proteasomes; Proteins; Recombinant protein expression; Sugar; Tobacco; Glycan array; Carbohydrate-binding; Fbs; Plant lectin; LacNAc; Nictaba; Lewis structure; Recombinant protein expression; LacNAc, N-acetyllactosamine; Nictaba, Nicotiana tabacum agglutinin; Fbs, sugar-binding F-box protein
• ISSN: 2211-5463; 2211-5463
• DOI: 10.1016/j.fob.2012.06.002

▸ Recombinant expression of a new type of Arabidopsis F-box protein. ▸ Arabidopsis F-box-Nictaba protein is a functional lectin. ▸ Arabidopsis F-box-Nictaba recognizes N-acetyllactosamine and Lewis structures.
The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.