Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris.
Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.
Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, 9000 Ghent, Belgium.
Bassam
B
Al Atalah
Pierre
P
Rougé
David F
DF
Smith
Paul
P
Proost
Yi
Y
Lasanajak
Els J M
EJ
Van Damme
D015870:Gene Expression; D037601:Mannose-Binding Lectin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D012275:Oryza / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010843:Pichia; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D037601:Mannose-Binding Lectin; D010940:Plant Proteins; D011994:Recombinant Proteins