Non‐branched β‐1,3‐glucan oligosaccharides trigger immune responses in Arabidopsis

[EN] Fungal cell walls, which are essential for environmental adaptation and host colonization by the fungus, have been evolutionarily selected by plants and animals as a source of microbe-associated molecular patterns (MAMPs) that, upon recognition by host pattern recognition receptors (PRRs), trigger immune responses conferring disease resistance. Chito-oligosaccharides [b-1,4-N-acetylglucosamine oligomers, (GlcNAc)n] are the only glycosidic structures from fungal walls that have been well-demonstrated to function as MAMPs in plants. Perception of (GlcNAc)4–8 by Arabidopsis involves CERK1, LYK4 and LYK5, three of the eight members of the LysM PRR family. We found that a glucan-enriched wall fraction from the pathogenic fungus Plectosphaerella cucumerina which was devoid of GlcNAc activated immune responses in Arabidopsis wild-type plants but not in the cerk1 mutant. Using this differential response, we identified the non-branched 1,3-b-D-(Glc) hexasaccharide as a major fungal MAMP. Recognition of 1,3-b-D-(Glc)6 was impaired in cerk1 but not in mutants defective in either each of the LysM PRR family members or in the PRR-co-receptor BAK1. Transcriptomic analyses of Arabidopsis plants treated with 1,3-b-D-(Glc)6 further demonstrated that this fungal MAMP triggers the expression of immunity-associated genes. In silico docking analyses with molecular mechanics and solvation energy calculations corroborated that CERK1 can bind 1,3-b-D-(Glc)6 at effective concentrations similar to those of (GlcNAc)4. These data support that plants, like animals, have selected as MAMPs the linear 1,3-b-D-glucans present in the walls of fungi and oomycetes. Our data also suggest that CERK1 functions as an immune co-receptor for linear 1,3-b-D-glucans in a similar way to its proposed function in the recognition of fungal chito-oligosaccharides and bacterial peptidoglycan MAMPs.S

Arabidopsis immune responses triggered by cellulose‐ and mixed‐linked glucan‐derived oligosaccharides require a group ofleucine‐rich repeat malectinreceptor kinases

[EN] The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern-triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked b- 1,3/b-1,4-glucans (MLGs; e.g. b-1,4-D-(Glc)2-b-1,3-D-Glc, MLG43) and cellulose (e.g. b-1,4-D-(Glc)3, CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. b-1,4-D-(GlcNAc)6, CHI6), which involve several receptor kinase proteins (RKs) with LysM-ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1–igp4 are altered in three RKs – AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) – with leucine-rich-repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T-DNA insertional loss-of-function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECDRKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD = 1.19 0.03 lM) and cellopentaose (KD = 1.40 0.01 lM), but not to MLG43, supporting its function as a plant PRR for cellulose-derived oligosaccharides. Our data suggest that these LRR-MAL RKs are components of a recognition mechanism for both cellulose- and MLG-derived oligosaccharide perception and downstream PTI activation in Arabidopsis.SIGrant PID-2021-126006OB-100 from the Spanish Ministry of Science and Innovation to AMThis work has also been financially supported by the ‘Severo Ochoa (SO) Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigaci on (AEI) of Spain (grants SEV-2016-0672 (2017-2021) and CEX2020-000999-S (2022-2025) to the CBGP). In the frame of the SO program, HM and PF-C were supported with postdoctoral fellowships. MM-D, DJB and DR were recipients of PhD Fellows PRE2019-088120 and PRE2019-091276 (SEV-2016- 0672) from AEI, and IND2017/BIO-7800 from Madrid Regional Government, respectively. Research in the lab of JS was financially supported by the University of Lausanne, the European Research Council (ERC) (grant agreement no. 716358) and the Swiss National Science Foundation (grant no. 310030_204526)

A computational structural study on the DNA-protecting role of the tardigrade-unique Dsup protein

18 Pág.The remarkable ability of tardigrades to withstand a wide range of physical and chemical extremes has attracted a considerable interest in these small invertebrates, with a particular focus on the protective roles of proteins expressed during such conditions. The discovery that a tardigrade-unique protein named Dsup (damage suppressor) protects DNA from damage produced by radiation and radicals, has raised expectations concerning its potential applications in biotechnology and medicine. We present in this paper what might be dubbed a "computational experiment" on the Dsup-DNA system. By means of molecular modelling, calculations of electrostatic potentials and electric fields, and all-atom molecular dynamics simulations, we obtained a dynamic picture of the Dsup-DNA interaction. Our results suggest that the protein is intrinsically disordered, which enables Dsup to adjust its structure to fit DNA shape. Strong electrostatic attractions and high protein flexibility drive the formation of a molecular aggregate in which Dsup shields DNA. While the precise mechanism of DNA protection conferred by Dsup remains to be elucidated, our study provides some molecular clues of their association that could be of interest for further investigation in this line.B.C.Z. acknowledges Universidad Politécnica de Madrid and Banco Santander for a predoctoral Programa Propio grant. M.G.A. received fund support from AllerScreening Project EU SEP-210415617 from European Commission, Horizon 2020 Programme. All molecular dynamics calculations were carried out on the Magerit supercomputer of Universidad Politécnica de Madrid. The authors acknowledge the computer resources and technical assistance provided by the Centro de Supercomputación y Visualización de Madrid (CeSViMa). The authors also want to acknowledge the "Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672, 2017-2021) for supporting the scientific services used in this work.Peer reviewe

Dynamic plasticity of the lipid antigen-binding site of CD1d is crucially favoured by acidic pH and helper proteins

16 Pág.CD1 molecules present lipid antigens for recognition by T-cell receptors (TCRs). Although a reasonably detailed picture of the CD1-lipid-TCR interaction exists, the initial steps regarding lipid loading onto and exchange between CD1 proteins remain elusive. The hydrophobic nature of lipids and the fact that CD1 molecules are unable to extract lipids from membranes raise the need for the assistance of helper proteins in lipid trafficking. However, the experimental study of this traffic in the endosomal compartments at which it occurs is so challenging that computational studies can help provide mechanistic insight into the associated processes. Here we present a multifaceted computational approach to obtain dynamic structural data on the human CD1d isotype. Conformational dynamics analysis shows an intrinsic flexibility associated with the protein architecture. Electrostatic properties together with molecular dynamics results for CD1d complexes with several lipids and helper proteins unravel the high dynamic plasticity of the antigen-binding site that is crucially favoured by acidic pH and the presence of helper proteins.This research was funded by Spanish Ministerio de Ciencia e Innnovación, Grant Number BIO2017–84548R and Comunidad de Madrid, Project FOODAL S2018/BAA-4574. B.C.Z. acknowledges Universidad Politécnica de Madrid and Banco Santander for a predoctoral Programa Propio grant. M.G.A. received fund support from AllerScreening Project EU SEP-210415617 from European Commission, Horizon 2020 Programme. All molecular dynamics calculations were carried out on the Magerit supercomputer of Universidad Politécnica de Madrid. The authors acknowledge the computer resources and technical assistance provided by the Centro de Supercomputación y Visualización de Madrid (CeSViMa). The authors also want to acknowledge the “Severo Ochoa Program for Centres of Excellence in R&D” from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672, 2017-2021) for supporting the scientific services used in this work.Peer reviewe

Structural Dynamics of the Lipid Antigen-Binding Site of CD1d Protein

22 Pág.CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1‒lipid‒T-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid-antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.This research was funded by Spanish Ministerio de Ciencia e Innovación, grant number BIO2017-84548R and Comunidad de Madrid, Project FOODAL S2018/BAA-4574. B.C.Z. received fund support from a predoctoral Programa Propio grant, Universidad Politécnica de Madrid and Banco Santander. M.G.A. received fund support from AllerScreening project EU SEP-210415617 from European Commission, Horizon 2020 Programme.Peer reviewe

The Role of Sphingolipids in Allergic Disorders

12 Pág.Allergy is defined as a complex chronic inflammatory condition in which genetic and environmental factors are implicated. Sphingolipids are involved in multiple biological functions, from cell membrane components to critical signaling molecules. To date, sphingolipids have been studied in different human pathologies such as neurological disorders, cancer, autoimmunity, and infections. Sphingolipid metabolites, in particular, ceramide and sphingosine-1-phosphate (S1P), regulate a diverse range of cellular processes that are important in immunity and inflammation. Moreover, variations in the sphingolipid concentrations have been strongly associated with allergic diseases. This review will focus on the role of sphingolipids in the development of allergic sensitization and allergic inflammation through the activation of immune cells resident in tissues, as well as their role in barrier remodeling and anaphylaxis. The knowledge gained in this emerging field will help to develop new therapeutic options for allergic disorders.This research was funded by the Spanish Government (MINECO, grant BIO2017-84548- R); by Instituto de Salud Carlos III (ISCIII) (PI19/00044); by ISCIII co-founded by FEDER Thematic Networks and Cooperative Research Centres: ARADYAL (RD16/0006/0003; RD16/0006/0015); and by Community of Madrid (FOODAL-CM_S2018/BAAA-4574). JT-A was funded by Instituto de Salud Carlos III (ISCIII), co-founded by FEDER Thematic Networks and Cooperative Research Centres: ARADYAL (RD16/0006/0003).Peer reviewe

Statistical Study of Low-Intensity Single-Molecule Recognition Events Using DeepTipTM Probes: Application to the Pru p 3-Phytosphingosine System

13 Pág.The interaction between the plant lipid transfer protein Pru p 3 and phytosphingosine was assessed using an atomic force microscope. Phytosphingosine was covalently immobilized on DeepTipTM probes and Pru p 3 on MicroDeckTM functionalized substrates. Single-molecular interaction events between both molecules were retrieved and classified and the distribution for each one of the identified types was calculated. A success rate of over 70% was found by comparing the number of specific Pru p 3-phytosphingosine interaction events with the total number of recorded curves. The analysis of the distribution established among the various types of curves was further pursued to distinguish between those curves that can mainly be used for assessing the recognition between phytosphingosine (sensor molecule) and Pru p 3 (target molecule) in the context of affinity atomic force microscopy, and those that entail details of the interaction and might be employed in the context of force spectroscopy. The successful application of these functionalized probes and substrates to the characterization of the low-intensity hydrophobic interaction characteristic of this system is a clear indication of the potential of exploiting this approach with an extremely wide range of different biological molecules of interest. The possibility of characterizing molecular assembly events with single-molecule resolution offers an advantageous procedure to plough into the field of molecular biomimetics.This study was partially funded by the Ministerio de Ciencia e Innovación (PID2020-116403RB-I00; MCIN/AEI/10.13039/501100011033) and by the Comunidad de Madrid (Spain) (MINA-CM P2022-BMD-7236, PIN1-00011.2/2022_INVESTIGO_CM, Tec4Bio-CM/P2018/NMT-4443 and PEJ-2021-AI/IND-21188). This work was funded by the European Union’s EIC-Pathfinder Programme, under the project THOR (Grant Agreement number 101099719). D.C.-O. wish to thank the support offered by the “Programa Propio de I + D + I para contratos predoctorales” by Universidad Politécnica de Madrid in association with Santander Universidades by Santander Bank.Peer reviewe

Unraveling the Diagnosis of Kiwifruit Allergy: Usefulness of Current Diagnostic Tests

Objectives: To determine the usefulness of the in vitro and in vivo methods used in the diagnosis of kiwifruit allergy and to specifically assess the impact of seed proteins on sensitivity. Methods: We performed skin prick tests (SPTs) using various commercial extracts, homemade pulp, and seed extracts and prick-prick tests with kiwifruit on 36 allergic patients. The presence of specific IgE (sIgE) was assessed using the ImmunoCAP (kiwifruit extract), ELISA (Act d 1, Act d 2), ISAC, and FABER assays. Immunoblotting of seed extract was carried out, and a single-blind oral food challenge was performed with whole seeds in seed-sensitized individuals. Results: The prick prick test with kiwifruit demonstrated the highest diagnostic capacity (81.8% sensitivity and 94.1% specificity) among the in vivo tests. The sIgE levels measured using ImmunoCAP (kiwifruit extract) showed a similar sensitivity to that of global ISAC and FABER (63.9%, 59.5%, and 58.3%, respectively). Act d 1 was the major allergen. Sensitization to Act d 1 was associated with positive sIgE results to whole kiwifruit extract detected by ImmunoCAP (P<.000). A positive SPT result to kiwifruit seeds was associated with severe symptoms induced by kiwifruit (P=.019) as a marker of advanced disease, but not with clinically relevant sensitization. Challenge testing with kiwifruit seeds performed on 8 seed-sensitized patients yielded negative results. Conclusions: Sensitization to Act d 1 is associated with a positive result in conventional diagnostic techniques, whereas kiwifruit seed sensitization does not increase the sensitivity of the diagnostic techniques evaluated.Objetivos: Determinar la rentabilidad diagnóstica de las técnicas in vitro e in vivo utilizadas en el diagnóstico de alergia al kiwi y estudiar la influencia de las proteínas alergénicas de las semillas en su sensibilidad. Métodos: Se seleccionaron 36 pacientes alérgicos a kiwi. Se les realizó prick test con cuatro extractos comerciales diferentes y prick-prick con kiwi. Se determinó IgE específica mediante ImmunoCAP (extracto de kiwi), ELISA (Act d 1, Act d 2), las micromatrices ISAC y FABER e Immunoblotting de extracto de semilla de kiwi. Se realizó exposición oral simple ciego frente a semilla de kiwi en pacientes sensibilizados a la semilla. Resultados: El prick-prick de kiwi fue la prueba in vivo con mayor rendimiento (sensibilidad 81,8%, especificidad 94,1%). El ImmunoCAP de extracto de kiwi mostró una sensibilidad similar a la global del ISAC y del FABER (63,9%, 59,5% y 58,3%, respectivamente). Act d 1 fue el alérgeno mayoritario. Se encontró asociación entre los niveles de IgE específica frente a Act d 1 (ISAC) y el extracto de kiwi mediante ImmunoCAP (p <0,000). La prueba cutánea positiva con semilla se asoció con mayor gravedad de síntomas frente a kiwi (p = 0,019), como marcador de enfermedad avanzada, pero no como sensibilización clínicamente relevante. La prueba de provocación con semillas fue negativa en los ocho pacientes provocados. Conclusiones: La sensibilización a Act d 1 se asocia con resultados positivos con las técnicas diagnósticas convencionales. La sensibilización frente a semillas no mejora el rendimiento de las técnicas evaluadas