Lig v 1 structure and the inflammatory response to the Ole e 1 protein family
Authors: Tania Robledo Retana, Jack Bradley‐Clarke, Tristan Croll, Ruth Rose, Inva Hoti, Andrew J. Stagg, Mayte Villalba, Richard W. Pickersgill
Journal: Allergy (2020)

To the Editor, Pollen grains of the Oleaceae family are a widespread source of respiratory allergens.1 The major allergen from these pollen grains is Ole e 1 homologues, including Ole e 1 (olive), Fra e 1 (ash), Lig v 1 (privet) and Syr v 1 (lilac) with identity among them above 83%. Additionally, other species produce members of this protein family with an average 30% identity with Oleaceae allergens, including Pla l 1 (English plantain), Che a 1 (lamb's quarter), Phl p 11 (Timothy grass) and Sal k 5 (Russian thistle).1-3 The Ole e 1 protein family promotes the production of specific IgE causing the development of type I allergies towards various pollen sources thanks to cross-reactivity. To date, 16 Ole e 1-like proteins are reported as allergens.2 We have determined the crystal structure of the major allergen from privet3 Lig v 1 (PDB 6YOA), which unexpectedly forms a homodimer (Figure 1A,B). Recombinant Lig v 1.0102 (rLig v 1) was produced as a glycosylated, non-tagged soluble protein in Pichia pastoris with the vector pPICZα/LIG13; it was purified and characterized before and after enzymatic de-glycosylation with PNGase F. Protein crystals were grown with pure rLig v 1 and analysed with X-rays; additionally, it was immunodetected by WB with anti-Fraxinus polyclonal antibodies4 and IgE from patients serum (Figure S1). CD spectrophotometry revealed Lig v 1 comprised 40% β-sheet and 60% irregular structure and revealed the protein was folded and thermostable up to 60°C (methods and results at Supporting Information). The structure of the homologue Pla l 1 (PDB 4Z8W) showed the Ole e 1-like protein family comprises a 7-strand β-barrel fold stabilized by three conserved disulphide bonds.2 When the crystal structures of Lig v 1 dimer and Pla l 1 are compared, the β-barrel and three disulphides bonds are conserved (Figure S2). Additionally, rLig v 1 has a C-terminal α-helix and significant differences in the loop structures at the N-terminal end of the β-barrel (Figure 1E) and the dimerization interface is different from that proposed to lead to oligomerization in Pla l 1. This is the first structure of the C-terminal region which is helical, a region shared by the family paradigm Ole e 1 which is a major epitope, as are the N-terminal loops.5 The loops 3, 5 and 7 correspond to the connections between β2-β3 (residues 45-54), β4-β5 (residues 68-76) and β6-β7 (residues 107-119), respectively (Figure 1C,F). Loop 7 is the longest and has the greatest difference in conformation to the corresponding loop in Pla l 1. The determined Lig v 1 structure provides knowledge of both loop conformation and flexibility. Lig v 1 forms a dimer with approximately twofold symmetry in the crystal (Figure 1A,B), but it is unclear if this is of functional significance. Ole e 1 subunits can polymerize and form dimers with higher allergenicity.6 Comparison of the structure of the loops between the two copies in the crystal reveals striking divergence in the conformation of the loops, highlighting the high flexibility and conformational heterogeneity present in the loop structures of the Ole e 1 family (Figure 1F,G). Although the residue Asn111 is glycosylated (Figure S1), there is little evidence of glycosylation in the electron density map because this residue is in a highly solvent-exposed position that provides no steric constraint in the crystal on the orientation of flexibility of the carbohydrate. The biological function of the Ole e 1 family proteins is unknown and the search for similar structures to Lig v 1 using DALI server found Pla l 1 as the most similar. Lower sequence identities were detected with fibrinogen binding protein from Staphylococcus aureus (4JDZ), and the collagen adhesion protein from the major pilin subunit of Bacillus cereus (3RKP, 3RKT), at 16% and 14%, respectively. The structural similarities include a central barrel connected by loops, consistent with a binding function, supporting the proposal that the Ole e 1 family orchestrate pollen tube assembly. A review of the literature suggests Ole e 1 is involved in pollen tube development and Pla l 1 is proposed to have a role in the modification of the cell wall in the extracellular space. The initiation of the pollen tube development starts with calcium flux into the pollen grain and it is likely that the Ole e 1 proteins interact with carbohydrates in the plant cell and orchestrate pollen tube growth, or they may simply fix the pollen grain in position while the pollen tube grows which is consistent with polymerization proposed for Pla l 1.2, 6 Recent studies showed the surfactant activity of Ole e 1 on epithelial cells7 suggesting the biology of Ole e 1-proteins could influence the development of allergy. Hence, we aimed to establish the effect of Lig v 1 in the activation of the inflammatory response by the innate immune system in the second part of this work. Peripheral blood mononuclear cells (PBMCs) isolated from randomly selected healthy donors were stimulated with rLig v 1 for 3 hours, detailed methods and analyses are described in Supporting Information. In brief, cells were stained with fluorochrome-conjugated monoclonal antibodies to surface molecular markers (HLA-DR, CD14, and CD16), and intracellularly stained for TNFα (flow cytometry experiments) or NF-kβ (co-localization experiments). FACS data were analysed with WinList software (version 7.1; Verity Software House), and NF-kβ was analysed with IDEAS software (Amnis Co.), using the co-localization wizard. HLA-DR+ cells were gated into classical (CD14+ CD16−), intermediate (CD14+ CD16+) and non-classical (CD14+/− CD16+) monocyte subsets. Production of TNFα and NF-kβ localization were assessed for each monocyte population. Cell incubation with Lig v 1 resulted in translocation of NF-kβ into the nucleus (Figure 2D) and the production of TNFα when compared to non-stimulated cells. Importantly, TNFα production was maintained or enhanced in the presence of the specific TLR4 inhibitor CLI-095 (Figure 2A), inhibitors MyD88-dependent TLR signalling (Figure 2B), or TRIF-dependent TLR signalling (Figure 2C) after rLig v 1 stimulation. In contrast, TNFα production stimulated by the TLR4 agonist Escherichia coli LPS (endotoxin) was completely blocked by both inhibitors as expected (Figure 2). Therefore, monocyte activation by rLig v 1 is TLR independent and cannot be attributed to endotoxin contamination. Activation with Lig v 1 also induced secretion of IL-10 (Figure 2E), IL-1β (Figure 2F), TNFα (Figure 2G) and IL-6 (Figure 2H) after stimulation of PMBCs with Lig v1 for 6 or 24 hours, although IL-33 and IL-12p(70) were not detected (Supporting Information). The activation of innate immune cells independently of TLRs has been previously reported for other allergens,8, 9 but this is the first time this pathway has been explored with the Ole e 1 family proteins. These results show for the first time the ability of Lig v 1 to activate innate immune cells. To understand better the contribution of these effects to allergy, it would be interesting in future to compare Lig v 1 with other Ole e 1-like proteins and to analyse responses from allergic individuals as well as healthy donors. Dr Terán and the lab of Immunogenetics and Allergy, from the National Institute of Respiratory Diseases (INER, Mexico) for the pooled serum from monosensitized patients. Dr Mohamed Ali Pereyra Morales, Biochemistry Department, Medicine Faculty, National Autonomous University of Mexico (UNAM) for the rabbit polyclonal serum anti-Fraxinus allergens. Professor Daniel J. Pennington of the Centre of Immunobiology, Blizard Institute, Queen Mary University of London for helpful discussions. Diffraction data were collected at the Diamond Light Source (beamline i03). The authors declare that they have no conflicts of interest. This work was supported by a CONACYT scholarship (TRR: 409358), Biotechnology and Biological Sciences Research Council LIDo DTP (JB-C), Wellcome Trust (CT: 209407/Z/17/Z), and by Queen Mary University of London. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.