ABSTRACT
Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma.
Subject(s)
Allergens/immunology , Cats/immunology , Glycoproteins/immunology , Lipopolysaccharides/immunology , Respiratory Hypersensitivity/immunology , Allergens/chemistry , Animals , Cells, Cultured , Cytokines/biosynthesis , Dogs , Flagellin/immunology , Glycoproteins/chemistry , Glycosylation , Granulocytes/immunology , Granulocytes/metabolism , Humans , Immunity, Innate , Ligands , Lipocalins/immunology , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/immunology , Lipopolysaccharides/metabolism , Lymphocyte Antigen 96/genetics , Lymphocyte Antigen 96/immunology , Lymphocyte Antigen 96/metabolism , Macromolecular Substances , Macrophages/immunology , Macrophages/metabolism , Mice , Models, Immunological , Protein Binding , Protein Processing, Post-Translational , Recombinant Fusion Proteins/immunology , Respiratory Hypersensitivity/etiology , Species Specificity , Specific Pathogen-Free Organisms , Structure-Activity Relationship , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/immunology , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , TransfectionABSTRACT
Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.
Subject(s)
Phospholipase C gamma/chemistry , rac GTP-Binding Proteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Activation , Epidermal Growth Factor/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Phospholipase C gamma/metabolism , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , Thermodynamics , rac GTP-Binding Proteins/metabolism , RAC2 GTP-Binding ProteinABSTRACT
Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.
