Arabidopsis F‐box protein containing a Nictaba‐related lectin domain interacts with N ‐acetyllactosamine 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.