Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris

Abstract

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.

Evolutionary relationships and expression analysis of EUL domain proteins in rice (Oryza sativa)

“…However, some discrepancies occurred according to the number and length/strength of hydrogen bonds anchoring the sugars to the carbohydrate-binding site. The docking experiments with OsEULS2 confirmed the previously reported substrate specificity for high-mannose N-glycans and lactosamine related structures (Al Atalah et al 2012). However, for OsEULD1A a substrate specificity for galactose related sugars or galactose containing glycoproteins was reported whereas mannose did not inhibit the agglutination of rabbit erythrocytes caused by OsEULD1A.…”

Lectin domains at the frontiers of plant defense

“…Similarly, both EUL domains composing the two-domain EUL protein from rice, OrysaEULD1A, show carbohydrate specificity toward galactose containing glycans (Al Atalah et al, 2014b). In contrast, the rice protein OrysaEULS2 preferably binds mannosylated N-glycan structures (Al Atalah et al, 2012). All these EUL proteins are located in the nucleus and the cytoplasm of the plant cell.…”

Promiscuity of the Euonymus Carbohydrate-Binding Domain

“…All residues forming the putative carbohydrate-binding site of the EUL domain are extremely conserved (Figure 2) and putative carbohydrate-binding sites appear as a charged groove as shown from the mapping of the electrostatic potentials on the molecular surface of the EUL domains [28]. An aromatic residue located in the vicinity of the putative carbohydrate-binding site e.g.…”

“…In contrast to EEA, which is expressed at relatively high levels in the arilli of the spindle tree seeds (500 µg/g of dry arillus material), the EUL proteins from Arabidopsis , rice and Physcomitrella are very low abundance proteins. Therefore the EUL domains of ArathEULS3, OrysaEULS2 and PhypaEULS3 were recombinantly expressed in the heterologous expression system Pichia pastoris and the recombinant proteins purified [ 18 , 28 ].…”

“…The agglutination of rabbit erythrocytes by the recombinant OrysaEULS2 protein from rice was best inhibited by mannose, methyl α- mannopyranoside and lactose as well as the glycoproteins thyroglobulin, ovomucoid and asialomucin [ 28 ]. In contrast mannose and methyl α-mannopyranoside did not inhibit the agglutination of rabbit erythrocytes by the recombinant PhypaEULS3 from the lower plant Physcomitrella .…”