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Mol Biol Rep (2013) 40:681–686 Structural biology of mite allergens Received: 1 August 2012 / Accepted: 3 October 2012 / Published online: 9 October 2012Ó Springer Science+Business Media Dordrecht 2012 Mite allergens contribute to a significant pro- of allergens The production of therapies against these portion of human allergic symptoms, including asthma and allergens, to alleviate or eliminate allergic symptoms, rhinitis. The development of therapies to treat and prevent hinges on our understanding of the individual allergens these symptoms depends largely on our understanding of produced by dust mites as well as by storage mites like the properties of these allergens. Much effort has been Blomia tropicalis. In fact, many studies have been devoted devoted to determining the structure and organization of to understanding the D. farinae and D. pteronyssinus mite allergens, particularly of the house dust mites, toward allergens from clinical, molecular, and immunological understanding their activities and how they elicit immu- standpoints. In particular, analysis of allergen protein nological responses in humans. Here, we review the structure and organization has advanced the field so far as structural biology of the major allergens from two species to allow the production of recombinant allergens. Impor- of house dust mites, Dermatophagoides farinae and tantly, allergen engineering offers the potential for large- D. pteronyssinus, as well as allergens from a storage mite, scale production of more effective treatments, therefore Blomia tropicalis. The knowledge gained from the struc- such studies are indispensible in reducing allergic disease.
tural biology of these allergens will enable progress in Studies of the structural biology of allergens, including producing novel, more effective treatments for mite aller- Dermatophagoides, have established the foundations on gies based on specific immunotherapy approaches.
which to explore the properties that determine allergen-icity. Beginning with amino acid sequence homology, the Mite
Allergen
Structural biology higher-order structure and organization of the proteins canbe derived to determine IgE binding capabilities or predictfunctions. Such information can also be used to understand potential cross-reactivity with other allergens. Thus, thecontributions of structural biology approaches to study- Dust and storage mites produce allergens leading to ing mite allergens are crucial to advancing both clinical widespread and common allergic diseases, including bron- and immunological perspectives on these disease-causing chial asthma, perennial rhinitis, and atopic dermatitis, that affect about 10–20 % of the global population Two dust mite species, Dermatophagoides farinae andD. pteronyssinus, are the predominant sources of these Structural biology: the means to an end allergens. Indeed, these species produce at least 23 groups Structural biology studies seek to determine the physicalproperties of protein organization as a basis for imputing functions and interactions of a protein. These kinds of stud- Department of Laboratory Medicine, Yancheng Health ies are commonly employed in allergen research because of Vocational & Technical College, Jiefangnan Road 263, the ease with which protein databases allow rapid classi- Yancheng 224006, Jiangsu Province, People's Republic of Chinae-mail: [email protected] fication and comparison of suspected allergens with known Mol Biol Rep (2013) 40:681–686 proteins, particularly other allergens. Ascertaining how the atomic level, allowing the definition of tertiary and quater- protein folds allows researchers to deduce whether/to what nary organization (in fact, primary and secondary structure extent the protein binds IgE antibodies; uncovering the can be determined at the same time). However, each of these size, stability, and solubility of an allergen aids in calcu- approaches carries disadvantages. X-ray crystallography lating its ability to cross mucosal barriers. Thus, structural consumes time and materials and cannot offer insight to information for allergens helps researchers gauge both the protein dynamics. NMR, while providing information on allergenicity—whether a protein induces a clinical or protein dynamics, is limited by the size of the protein being immunological allergic response—and cross-reactivity— studied; larger macromolecules are precluded. Additionally, ability to bind the same epitope as other proteins—of these the comparability of crystal structures and solution-based structures (as in NMR) remains somewhat controversial.
A variety of techniques are used to determine the Despite these drawbacks, both approaches are widely used structure of a protein. From sequence homology searches and continue to offer invaluable information toward the and computer modeling, to X-ray crystallography and understanding of protein structure, function, and interac- nuclear magnetic resonance spectroscopy, the availability tions. Further, these methods have enabled the classification of both virtual and experimental approaches provides a of many different allergens and promoted a better under- range of possible methods to gain insight to protein orga- standing of allergic disease.
nization and function. Indeed, the recent windfall of novel Finally, additional virtual methods are used to uncover proteins—including allergens [, ]—identified through biophysical information about proteins like allergens. Pro- large-scale proteomics studies has placed structural biology grams like Visual Molecular Dynamics (VMD; techniques at the forefront of characterizing these new ), and the related NAMD, allow researchers to simulate the interactions of proteins, including The primary structure of a protein is determined by amino their abilities to bind or cross membranes. The ability to acid sequencing using standard molecular biology tech- model allergens in silico can lead to discoveries regarding niques, and often includes extrapolation from the nucleic cross-reactivity or allergenicity, as has been done for acid sequence of a gene. Additionally, advanced mass spec- severity of response to peanut allergens [].
trometry methods are commonly used to determine theamino acid composition of proteins. Often, though, bioin-formatics analyses form the foundation for further charac- Structural biology of mite allergens terization. Many freely-accessible databases (e.g., NCBI,PDB) are available for sequence alignments to identify The application of structural biology to the mite allergens similarities to proteins from other species or those with has advanced both clinical and immunological under- defined functions. Alignments also aid in predicting post- standings of how these proteins promote allergic disease.
translational modifications that can alter a protein's confor- Allergens from Dermatophagoides are classified based on mation or activity.
immune response into major groups 1 and 2 and middle The characterization of proteins beyond primary struc- groups 4, 5, and 7 However, there are at least 23 groups ture becomes gradually more complicated. Secondary struc- of allergens from the two most common dust mite species, ture, or the pattern of arrangement encompassing strands, D. farinae and D. pteronyssinus, all of which seem to elicit helices, turns, and coils, while often predicted through some degree of immune response [, ]. The importance of computer modeling based on primary sequence, can be structural biology approaches to classifying and under- determined in solution using experimental approaches such standing these allergens has been invaluable—from out- as circular dichroism spectroscopy (CD). CD employs lining the basic properties of the proteins, to determining optical absorbance to determine the conformation of a the ability to bind IgE and predicting cross-reactivity with protein in solution. Using this method, the arrangement of related allergens, and, finally, aiding in the development of a-helices, b-sheets, turns, and coils can be determined.
immunotherapy against the allergens Indeed, combin- While low in resolution, the application of CD can quickly ing what is known about the major allergen groups with and reliably identify the secondary structure of a protein.
what can be derived from related proteins described in For example, following primary structure determination existing databases, much more information will be forth- through mass spectrometry, the secondary structure of the coming regarding other allergen groups.
birch pollen allergen, Bet v 1, was deduced using CD The spatial structure has been determined for several of Higher-order structures are determined by more advanced the allergens of Dermatophagoides and B. tropicalis. Initial methods with higher resolution, particularly X-ray crystal- studies used in the late 1980s and early 90s used tradi- lography and nuclear magnetic resonance spectroscopy tional molecular and immunological approaches to purify (NMR). These techniques resolve protein structures at the the allergen proteins (affinity chromatography), determine Mol Biol Rep (2013) 40:681–686 their characteristics (SDS-PAGE), and quantify allergen- structure followed by a C-terminal extended loop and is tightly icity (ELISA, radioimmunoassay) (e.g., [–Due to the anchored at two regions of the mature enzyme. The prosegment advent of the many more advanced and specific techniques binding loop, the substrate binding cleft surrounding catalytic described above, a wide range of approaches have been residue Cys35, and Asn53, the potential N-glycosylation site in employed to derive more detailed information about mature Der f 1, is located on the surface distant from the catalytic allergens. Below, we will review what is known about site and the prosegment. These two regions on the molecular structures for the groups 1, 2, 5, 7, and 13 allergens, and surface of the mature portion were blocked by the prosegments as how structural biology has contributed to the understanding major conformational IgE epitopes. The allergenicities of the of the allergenicity of these proteins.
proforms are less than those of the mature forms in a broadpopulation of patients. Further, recombinant mature Der p 1 and Group 1 allergens Der f 1 are similar to the natural types in their structures andallergenicities and retain proteolytic activities The group 1 allergens from D. pteronyssinus and D. farinae,Der p 1 and Der f 1, respectively, are major mite allergens, Group 2 allergens accounting for [50 % of IgE antibodies against total miteextract [First isolated following cDNA cloning, these The Der p 2 and Der f 2 allergens belong to the ML domain proteins are 82 % identical in sequence and belong to a lipid binding protein family, which also includes the Nei- family of papain-like cysteine proteases [The crystal mann-Pick type C2 (NPC2) proteins []. The crystal structures of both mature Der p 1 and Der f 1, as well as structures of these proteins have been determined, reveal- their recombinant prodomains, have been solved , ing anti-parallel beta strands and a hydrophobic cavity [ Interestingly, despite extensive sequence homology, mature ]. Similarities with the structures of NPC2 proteins Der p 1 and Der f 1 do not exhibit identical structures. Der suggest a potential role in lipid binding or transport.
p 1 is monomeric and exhibits a cysteine protease fold and a The overall tertiary fold of Der f 2 is that of two anti- magnesium binding site , ]. Der f 1 is also monomeric, parallel b-pleated sheets overlying each other. This fold is but lacks a metal binding site [Structural differences are characteristic of the immunoglobulin superfamily. There also found at the N-terminal and central loop regions, those are three disulfide bonds in Der f 2, forming covalent bonds sites that are exposed for surface binding. Additionally, Der between residues 8 and 119, 21 and 27, and 73 and 78 [ f 1 appears to have fewer polymorphisms (five known) ], which are critical to the IgE-binding capacity of Der f compared to Der p 1 (23 known) The prodomains of 2. Indeed, Takai et al. [extended initial findings of the these two allergens differ from the mature forms with importance of these disulfide bonds to the allergenicity of respect to secondary structure ].
Der f 2 [] by studying engineered recombinant pro- The structural differences between Der p 1 and Der f 1 teins with mutations in these bonds. Alterations in the likely result in differences in IgE binding. While these disulfide bonds of Der f 2 led to conformational changes allergens exhibit high cross-reactivity in humans that reduced its IgE binding capacity. Therefore, loosening antibody binding demonstrates a high degree of species of the rigid tertiary structure by elimination of key intra- specificity [, monoclonal antibodies raised against molecular interactions in the allergen molecule would be either species are *95 % species-specific. Four surface an effective strategy in allergen engineering for safe and patches differ between Der f 1 and Der p 1, and areas these effective allergen-specific immunotherapy ].
proteins have in common are the putative basis for cross- The similarities between Der f 2 and Der p 2 explain the reactivity between the two allergens. The Der f 1 and Der extensive cross-reactivity between these allergens [ p 1 structures show the presence of a conserved water Indeed, the majority of the residues [86 % homology binding site at one end and in the catalytic site at the other.
between Der f 2 and Der p 2 ] on the binding surface of the The binding of water molecules could be biologically group 2 allergens is conserved, allowing for similar IgE significant and may serve to catalyze the cysteine protease binding capacities ]. NMR and structural modeling activity of the allergen studies indicate two surface patches representing the IgE The proforms of both Der p 1 and Der f 1 exhibit less aller- binding epitopes [ genicty than do the mature and recombinant forms. The siteswhere the prosegments are anchored likely reflect the major IgE Group 5 allergens epitopes of the two allergens ]. Based on the crystal structuresof other cysteine proteases, molecular modeling of pro-Der f 1 The group 5 allergens from D. pteronyssinus and D. farinae was performed to demonstrate information on the location of show similarity to the group 5 allergen from another house IgE-binding sites blocked by the propeptides. To be more exact, dust mite species, B. tropicalis. Der p 5 and Blo t 5 are the Der f 1 prosegment consists of an N-terminal domain- 42 % identical in amino acid sequence [Additionally, Mol Biol Rep (2013) 40:681–686 the crystal structure of Der p 5 was recently solved, Immunotherapy against mite allergens revealing a monomeric protein that appears similar to thesolution structure of Blo t 5 ]. Further, Der p 5 has a Given the high allergenicity of the groups 1 and 2 allergens helical structure that seems to interact with itself in an anti- from house dust mites, effort has been devoted to developing parallel manner and a hydrophobic cavity. However, the specific immunotherapies (SIT) against these proteins.
function and activity of Der p 5 and other group 5 allergens Additionally, advances in generating large amounts of remain unknown.
recombinant proteins have led to animal-model studies of The group 5 allergens represent an important allergen potential vaccines. For example, the changes induced in IgE group because of high responsiveness of patients to dust binding capacity by altering the disulfide bonds of Der f 2 mite extracts containing these proteins. However, while suggests potential avenues for immunotherapy against the some cross-reactivity exists among the group 5 allergens, group 2 allergens. Studies of human cells in vitro and of in general IgE binding appears to be species-specific immunized mice indicate that the production of a hypoal- The Blo t 5 allergen appears to elicit IgE binding in 90 % lergen—an allergenic protein less capable of binding IgE but of asthmatic and allergic patients [However, further retaining potent T cell reactivity—through genetic tech- work is needed to understand the function and activity of niques may produce immunity to group 2 allergens [A this group of allergens.
vaccination was recently developed by creating hypoaller-gens using recombinant Der p 1 and Der p 2; testing in rabbits Group 7 allergens demonstrated good levels of efficacy and tolerability [Recent work also suggests that expressing the dominant T Group 7 allergens are fairly abundant in mite extracts.
cell epitopes of Der p 1 in transgenic rice seed may produce a Indeed, these are the 44th most abundant protein produced viable vaccination against group 1 allergens ].
by mites, ranking not far behind Der p 2 (41st) [These Several clinical trials have been conducted in recent allergens share sequence similarity to the prenylcysteine years to test the efficacy and safety of SIT against the dust lyase proteins predicted to exist in insects ]. Analyses mite allergens, albeit with mixed results. Two types of SIT also predict at least three isoforms exist for Der p 7; these have been used: subcutaneous (SCIT) and sublingual may represent different levels of glycosylation ]. A (SLIT). Some have used purified mite extracts; others have more recent characterization of Der p 7 indicates a struc- used mite extracts combined with adjuvant therapies. In ture comprising anti-parallel b sheets. Additionally, this general, it appears that these SIT against mite allergens are allergen appears to bind a bacterial lipid product and effective, but the magnitude of effect varies between resembles proteins in the Toll-like receptor pathway [ studies and types of SIT –A search of government- Some earlier work demonstrated that 50 % of dust mite- sponsored clinical trials in the U.S. (clinicaltrials.gov; allergic patients react to Der p 7. This protein also appears August 2012) returned 14 active trials investigating the to elicit a high T cell proliferation response []. This efficacy of house dust mite SIT. A windfall of findings specificity may be related to the similarity of Der p 7 to from these studies—and many more worldwide—can be Toll-like receptors, which are involved in innate immunity expected within the next several years.
[However, further work is necessary to uncover the While SIT still have far to go before widespread use in reason for this specificity.
humans, they offer some hope for eventual reduction ofallergic disease. Continued advances in the field will rely Group 13 allergens heavily on the information generated by structural biologystudies of each allergen. The work reviewed here has Group 13 allergens are considered fatty acid binding pro- established strong foundations for progress in both under- teins (FABP) and are fairly abundant in mite extracts standing the structural biology of allergens and developing NMR studies of Der f 13 revealed anti-parallel beta strands immunotherapies against them.
preceeded by a helix-turn-helix motif [Several surfaceresidues are conserved between group 13 allergens andother proteins involved in the transport of fatty acids, with the highest homology to human brain FABP.
Epitope mapping and mutational analysis indicate that Proteins produced by mite species, especially the Derma- the surface residues are critical to IgE binding. However, tophagoides, are responsible for allergic disease world- despite high abundance in mite extracts and good capacity wide. Knowledge gained from structural biology studies of to bing IgE, the group 13 allergens do not elicit a severe these allergens has established a foundation for develop- immune response; only some patients produce IgE against ment of new therapies to treat and prevent allergic disease.
these allergens [ Such studies will continue to contribute greatly to clinical Mol Biol Rep (2013) 40:681–686 advances, particularly in this era of high-throughput location and species specificity studied by solid-phase inhibition genomics and proteomics.
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