Cloning And Bioinformatics Analysis Of Major Allergens In Mollusks

Hypersensitive reactions triggered by molluscs are increasingly common in dailylife. Tropomyosin (TM) is considered to be a major allergen in molluscs. IgEcross-reactivity is clinically and experimentally recognized among molluscs and evenbetween crustaceans and mollusks. And it is reported that the ability to triggerhypersensitive reactions of tropomyosins from different species may differ much fromeach other, so as the subsequent allergic symptoms. Study on cross-reactivity amongdifferent species focus more on amino acid sequence similarity than structuresimilarity, while study on diversity of allergens is little. In this study, tropomyosinfrom Chlamys farreri was cloned by a cDNA cloning technique and bioinformaticswas used to analyze the amino acid sequence of tropomyosin to predict itssensitization. Meanwhile, allergens available now in mollusks were analyzed bybioinformatics approaches to analyze the allergen homologous and the sequencemicroheterogeneity, which is helpful to explore the cross-reactivity among molluscsand molecular differences among them for a further understanding of the scientificbasis of food allergies.1. The full-length cDNAs encoding tropomyosin from Chlamys farreri wascloned by RACE technology, whose cDNA contains an open reading frame composedof855bp coding for284amino acid residues. Among its amino acid composition,Tryptophan (W) along with Proline (P) and Cysteine (C) were absent. The molecularweight of TM from Chlamys farreri is32.5kDa, and isoelectric point is4.46. InDNAStar and AntheProt prediction system, five properties of the amino acid sequencewere chosen to be analyzed including secondary structure, hydrophilicity, flexibility,accessibility and antigenicity. The secondary structure was rich in alpha helix. And thelatter parameters indicate it likely to form epitopes. It can be seen from modelingresult that spacial structure of tropomyosin from Chlamys farreri was simple, with nocomplicated three or quaternary structure. Comprehensive results show that tropomyosin from Chlamys farreri is allergic one.2. Bioinformatics methods were used for homology analysis of allergens fromdifferent mollusks. Homology analysis indicated that the major allergens frommolluscs share high sequence similarity among them, ranging from65.1%to99.6%.Four conserved regions which contain at least six amino acid residues were found(120-125,164-178,214-228,251-261), and the region of251-261is located in thesixth epitope of Pen a1reported before,which is quietly possible the cross-reactiveIgE-binding epitopes among mollusks. Thus, this study is of particular value inmaking it possible to understand the overall features of the amino acid sequences ofTMs from edible molluscs.The analysis of secondary structures and sequence properties of tropomyosinsfrom molluscs were carried out by using methods of bioinformatics. Results showedthat the amino acid composition of all these molluscan tropomyosins lacks Proline (P),sharing close sequence of basic properties. Their secondary structures are mostlyalpha helix, and hydrophilicity and solvent accessibility are high. What is more,antigenic index of these tropomyosins is relatively high. It can be seen from modelingresult that spacial structures of tropomyosins from molluscs were completely simple,without complicated three or quaternary structures. Sharing common basic naturesand structure information makes it easier to understand the cross-reaction among themolluscs even with crustaceans and other shellfish.3. Bioinformatics methods were used for sequence microheterogeneity analysisof allergens from different mollusks. Tropomyosin sequences analyzed (18entries)are engaged into17unique cases of sequence microheterogeneity with sequenceidentity ranging from91.2%to99.6%. Examination of the distribution over alignedtropomyosin sequence of300cases of amino acid substitutions (affect90residuescomprising31.2%of the full sequence length) giving rise to sequencemicroheterogeneity.49.0%of the microheterogeneous substitutions (147of300substitutions) are classified as notably affecting protein stability, and31.0%(93of300substitutions) are located in the epitopes. What’ is more, among thosemicroheterogeneous substitutions, it appeals that Serine (S) and Alanine (A) is easier to be replaced. Sequence microheterogeneity of tropomyosins from different molluskswas explored for the first time, which provides a molecular basis for allergy diversitytriggerd by mollusks.In summary, full-length cDNAs encoding tropomyosin from Chlamys farreri wascloned and its structure with basic physical and chemical properties was predicted bymethods of bioinformatics. Moreover, tropomyosins available now in mollusks wereanalyzed by bioinformatics approaches to analyze the allergen homologous and thesequence microheterogeneity. The amino acids that easier to be replaced oftropomyosins from different molluscs were identified. All described above provides amolecular basis for allergy diversity triggerd by mollusks.