ABSTRACT
Octopus is an important mollusk in human dietary for its nutritional value, however it also causes allergic reactions in humans. Major allergens from octopus have been identified, while the knowledge of novel allergens remains poor. In the present study, a novel allergen with molecular weight of 28kDa protein was purified from octopus (Octopus fangsiao) and identified as triosephosphate isomerase (TIM) by mass spectrometry. TIM aggregated beyond 45°C, and its IgE-binding activity was affected under extreme pH conditions due to the altered secondary structure. In simulated gastric fluid digestion, TIM can be degraded into small fragments, while retaining over 80% of the IgE-binding activity. The full-length cDNA of O. fangsiao TIM (1140bp) was cloned, which encodes 247 amino acid residues, and the entire recombinant TIM was successfully expressed in Escherichia coli BL21, which showed similar immunoreactivity to the native TIM. Different intensity of cross-reactivity among TIM from related species revealed the complexity of its epitopes. Eight linear epitopes of TIM were predicted following bioinformatic analysis. Furthermore, a conformational epitope (A71G74S69D75T73F72V67) was confirmed by the phage display technology. The results revealed the physicochemical and immunological characteristics of TIM, which is significant in the development of hyposensitivity food and allergy diagnosis.
Subject(s)
Allergens/immunology , Epitopes, B-Lymphocyte/immunology , Octopodiformes/enzymology , Octopodiformes/immunology , Triose-Phosphate Isomerase/immunology , Adolescent , Adult , Allergens/chemistry , Allergens/genetics , Amino Acid Sequence , Animals , Child , Cloning, Molecular , Cross Reactions , Electrophoresis, Gel, Two-Dimensional , Epitope Mapping , Female , Food Hypersensitivity/etiology , Food Hypersensitivity/immunology , Humans , Immunoblotting , Male , Middle Aged , Models, Molecular , Octopodiformes/chemistry , Octopodiformes/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/genetics , Young AdultABSTRACT
BACKGROUND: Arginine kinase (AK) is expressed in a wide variety of species, including human food sources (seafood) and pests (cockroaches and moths), and has been reported as a novel allergen. However, there has been little research on the allergenicity of AK in crustaceans. In this study the physicochemical properties of AK from mud crab (Scylla paramamosain) were investigated. RESULTS: Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and inhibition enzyme-linked immunosorbent assay revealed that purified AK was unstable in thermal processing and in acid buffer. Under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, purified AK was much more readily degraded by pepsin than by trypsin or chymotrypsin. The unpurified AK in crab myogen degraded more markedly than purified AK. In addition, in two-phase gastrointestinal digestion, AK was rapidly degraded by pepsin but resistant to trypsin and chymotrypsin digestion, while tropomyosin derived from mud crab was resistant to pepsin digestion but digested readily by trypsin or chymotrypsin. Further study of serum samples obtained from crab-allergic human patients indicated that the allergenicity of AK was markedly reduced by digestion with SGF but not SIF. CONCLUSION: AK is an important food allergen despite its unstable physicochemical properties of digestibility.
Subject(s)
Allergens/chemistry , Arginine Kinase/chemistry , Arthropod Proteins/chemistry , Brachyura/chemistry , Shellfish/analysis , Allergens/adverse effects , Allergens/isolation & purification , Allergens/metabolism , Animals , Arginine Kinase/antagonists & inhibitors , Arginine Kinase/isolation & purification , Arginine Kinase/metabolism , Arthropod Proteins/antagonists & inhibitors , Arthropod Proteins/isolation & purification , Arthropod Proteins/metabolism , Brachyura/enzymology , Brachyura/growth & development , Chemical Phenomena , China , Dietary Proteins/analysis , Dietary Proteins/antagonists & inhibitors , Dietary Proteins/isolation & purification , Dietary Proteins/metabolism , Digestion , Enzyme Stability , Food Hypersensitivity/blood , Food Hypersensitivity/immunology , Gastric Juice/enzymology , Gastric Juice/metabolism , Hot Temperature , Humans , Hydrogen-Ion Concentration , Immunoglobulin E/metabolism , Mechanical Phenomena , Models, Molecular , Pepsin A/metabolism , Protein Structure, Tertiary , Proteolysis , Shellfish/adverse effectsABSTRACT
Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.
