Characterization of a type D1A EUL-related lectin from rice expressed in Pichia pastoris

Abstract

OrysaEULD1A is one of the five EUL genes in rice (Oryza sativa) encoding a putative carbohydrate-binding protein belonging to the family of Euonymus related lectins (EUL). The OrysaEULD1A sequence comprises two highly similar EUL domains (91% sequence similarity and 72% sequence identity) separated by a 23 amino acid linker sequence and preceded by a 19 amino acid N-terminal sequence. In the present study, the full-length protein OrysaEULD1A as well as its individual domains OrysaEULD1A domain 1 and 2 were expressed in Pichia pastoris. After purification of the recombinant proteins, their carbohydrate-binding specificity was analyzed and compared. Interestingly, all recombinant lectins showed clear specificity towards galactosylated structures. Furthermore, all recombinant proteins agglutinated red blood cells, indicating that the full-length protein OrysaEULD1A and its domains are true lectins. These results taken together with data previously reported for single-domain EUL proteins indicate that although the amino acids--responsible for the formation of the carbohydrate-binding site--are identical for all EUL proteins in rice, these lectins show different carbohydrate specificities. This promiscuity of the carbohydrate-binding site can be attributed to gene divergence.

OsEUL Lectin Gene Expression in Rice: Stress Regulation, Subcellular Localization and Tissue Specificity

“…OsEULD2 was significantly upregulated after Magnaporthe infection. These results are in agreement with a previous qPCR study using different time points which also showed the upregulation of OsEULD1b and OsEULD2 after Xanthomonas infection and the upregulation of OsEULD2 after disease by Magnaporthe (Al Atalah et al, 2014). Transcript levels for OsEULD2 were significantly upregulated by three different rice pathogens.…”

“…qRT-PCRs were performed using SYBR Green I Mix (Bio-Rad) and following conditions: 10 min initial denaturation step at 95°C, 42 cycles (15 s at 95°C, 25 s at 60°C, 20 s at 72°C) and detected with Bio-Rad CFX Connect™ Real-Time PCR Detection System. The expression of the genes was normalized against three reference genes: EXP (LOC_Os03g27010.1), EIF5C (LOC_Os011g21990.1), and EXP Narsai (LOC_Os07g02340.1) (Narsai et al, 2010;Kyndt et al, 2012;Al Atalah et al, 2014). The stability of the reference genes was checked by the qbase+ software (Biogazelle, Zwijnaarde, Belgium).…”

“…Despite the high sequence similarity between OsEULs the proteins have very different carbohydrate-binding activities. For instance, OsEULS2 has a preference for high-mannose N-glycans (Al Atalah et al, 2012) while OsEULD1a rather binds to galactose related sugars (Al Atalah et al, 2014). Analyses of the promoter region of the five rice EUL genes revealed a variety of stress responsive elements (De Schutter et al, 2017).…”

Lectin domains at the frontiers of plant defense

“…ArathEULS3 from A. thaliana preferentially interacts with N -glycans containing galactosylated structures such as Lewis X [Galβ1–4(Fucα1–3)GlcNAc], Lewis Y [Fucα1–2Galβ1–4(Fucα1–3)GlcNAc] and lactosamine (Galβ1–4GlcNAc) motifs (Van Hove et al, 2011). 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).…”

Overview of the Structure–Function Relationships of Mannose-Specific Lectins from Plants, Algae and Fungi

“…Besides Nictaba, which appears as the prototype of a group of closely related lectins [263,264], other groups of stress inducible lectins in the nucleocytoplasmic compartment have been identified, such as the Euonymus europaeus EUL-related lectins [170,265] and the group of mannose-binding jacalin-related lectins [221,266]. Among the stress inducible cytoplasmic/nuclear lectins identified so far, Nictaba belonging to the Nictaba-related lectins [267], Orysata belonging to the jacalin-related lectins [268], and OrysaEULD1A belonging to the EUL-related lectins [269], all readily interact with high-mannose glycan structures (Table 7). Accordingly, they participate as signaling molecules in the plant response to stress conditions [270].…”