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 (Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc) and poly-N-acetyllactosamine ([Gal beta 1-4GlcNAc](n)) as well as Lewis A (Gal beta 1-3(Fuc alpha 1-4) GlcNAc), Lewis X (Gal beta 1-4(Fuc alpha 1-3) GlcNAc, Lewis Y (Fuc alpha 1-2Gal beta 14(Fuc alpha 1-3) GlcNAc) and blood type B (Gal alpha 1-3(Fuc alpha 1-2) Gal beta 1-3GlcNAc) 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. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.