Discovery and phylogeny of a ricin-B-like domain from rice.

Lectins are carbohydrate-binding proteins which play key roles in various biological processes, including cell signaling, pathogen recognition and development. Previous research conducted on ricin-B lectin domains and carbohydrate-binding modules of family 13 (CBM13) illustrated the striking resemblances between these two groups of protein domains. In this study, we report on the discovery, identification and putative biochemical characteristics of a ricin-B-like domain that is unique for GH27 enzymes from land plants, identified in the OsAPSE enzyme from Japanese rice (Oryza sativa L. subsp. Japonica). HMM-BLAST against the CAZy database (E = 0.0091) indicated that this ricin-B-like domain is currently not classified in CAZy. BLAST retrieved 187 APSE homologs carrying a ricin-B-like domain, all from Embryophyta. Since the ricin-B-like domain contains only 65 amino acid residues it is remarkably shorter than the canonical CBM13/ricin-B domains of 120-130 residues. Across homologs, the ricin-B-like domain is always preceded by an N-terminal GH27 domain and in 91.2 % of proteins, the ricin-B-like domain is followed by a GH-all-beta domain at the C-terminus. Phylogeny and sequence similarity networks group ricin-B-like domains in clades distinct from the clades with CBM13/ricin-B domains. Structural alignments show limited overall similarity towards CBM13/ricin-B domains, with RMSD values between 4-5Å. Docking of carbohydrate structures to AlphaFold models indicate the presence of two putative carbohydrate-binding sites. Recombinant OsAPSE produced in E. coli, was purified, refolded and analyzed on a glycan microarray, but did not show any interaction with the glycans.