Crystal Structure of H227A Mutant of Arginine Kinase in Daphnia magna Suggests the Importance of Its Stability.

Arginine kinase (AK) plays a crucial role in the survival of Daphnia magna, a water flea and a common planktonic invertebrate sensitive to water pollution, owing to the production of bioenergy. AK from D. magna (DmAK) has four highly conserved histidine residues, namely, H90, H227, H284, and H315 in the amino acid sequence. In contrast to DmAK WT (wild type), the enzyme activity of the H227A mutant decreases by 18%. To identify the structure-function relationship of this H227A mutant enzyme, the crystal 3D X-ray structure has been determined and an unfolding assay using anilino-1-naphthalenesulfonic acid (ANS) fluorescence has been undertaken. The results revealed that when compared to the DmAK WT, the hydrogen bonding between H227 and A135 was broken in the H227A crystal structure. This suggests that H227 residue, closed to the arginine binding site, plays an important role in maintaining the structural stability and maximizing the enzyme activity through hydrogen bonding with the backbone oxygen of A135.

Physicochemical characterization and linear epitopes identification of arginine kinase allergen from Crassostrea gigas.

BACKGROUND: Molluscan shellfish, including oysters, often cause allergic reactions in sensitive people throughout the world. It has been demonstrated that arginine kinase (AK) is one of the major allergens of oyster. The present study aimed to evaluate the immunoreactivity and structure of oyster AK as affected by heat treatment, pH change, and in vitro digestion. What is more, the immunoglobulin E-binding epitopes of this allergen were also predicted and validated. RESULTS: Thermal and pH assays revealed that AK was unstable at temperature >40 °C or pH ≤5.0 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and circular dichroism, and the digestibility assays suggested that AK was more easily digested by pepsin than by trypsin and chymotrypsin. The potential epitopes were predicted through immunoinformatics tools, and seven linear epitopes were identified by indirect competition enzyme-linked immunosorbent assay with pooled sera and individual serum from oyster-allergic patients. The critical amino acids in each epitope were also confirmed using mutant peptides. These linear epitopes and critical amino acids were apt to distribute on the outer surface of homology-based AK model. Moreover, the three denaturants (sodium dodecyl sulfate, ß-mercaptoethanol, and urea) can destroy the spatial structure of AK and increase or reduce its allergenicity by denaturation treatments. CONCLUSION: Processing conditions lay the foundation for the variation of allergenicity. Seven linear epitopes and their critical amino acids were identified by indirect competitive enzyme-linked immunosorbent assay. These findings will be helpful in allergy diagnosis and development of hypoallergenic products in the near future. © 2021 Society of Chemical Industry.

Genomic structure, expression and functional characterization of arginine kinase (EcAK) from Exopalaemon carinicauda.

Arginine kinase (AK, EC 2.7.3.3) plays an important role in cells with high, fluctuating energy requirements. In invertebrates, AK is the major phosphagen kinase that modulates the energy metabolism. Here, the full-length cDNA sequence encoding arginine kinase (EcAK) was obtained from the Exopalaemon carinicauda. The complete nucleotide sequence of EcAK contained a 1068 bp open reading frame (ORF) encoding EcAK precursor of 355 amino acids. The genomic DNA fragment of EcAK with the corresponding cDNA sequence is composed of 4 exons and 3 introns. The domain architecture of the deduced EcAK protein contained an ATP-gua_PtransN domain and an ATP-gua_Ptrans domain. EcAK mRNA was predominantly expressed in the muscle. The expression of EcAK in the prawns challenged with Vibrio parahaemolyticus and Aeromonas hydrophila changed in a time-dependent manner. Then, EcAK was recombinantly expressed in Pichia pastoris and the purified recombinant EcAK had the same enzymatic characterization as AK from the muscle of Euphausia superba. In conclusion, EcAK may play the same biological activity in E. carinicauda as those from other crustaceans.

Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

Interaction of the Small GTPase Cdc42 with Arginine Kinase Restricts White Spot Syndrome Virus in Shrimp.

Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, ΔMjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.

Elevated µs-ms timescale backbone dynamics in the transition state analog form of arginine kinase.

Arginine kinase catalyzes reversible phosphoryl transfer between arginine and ATP. Crystal structures of arginine kinase in an open, substrate-free form and closed, transition state analog (TSA) complex indicate that the enzyme undergoes substantial domain and loop rearrangements required for substrate binding, catalysis, and product release. Nuclear magnetic resonance (NMR) has shown that substrate-free arginine kinase is rigid on the ps-ns timescale (average S2=0.84±0.08) yet quite dynamic on the µs-ms timescale (35 residues with Rex, 12%), and that movements of the N-terminal domain and the loop comprising residues I182-G209 are rate-limiting on catalysis. Here, NMR of the TSA-bound enzyme shows similar rigidity on the ps-ns timescale (average S2=0.91±0.05) and substantially increased µs-ms timescale dynamics (77 residues; 22%). Many of the residues displaying µs-ms dynamics in NMR Carr-Purcell-Meiboom-Gill (CPMG) 15N backbone relaxation dispersion experiments of the TSA complex are also dynamic in substrate-free enzyme. However, the presence of additional dynamic residues in the TSA-bound form suggests that dynamics extend through much of the C-terminal domain, which indicates that in the closed form, a larger fraction of the protein takes part in conformational transitions to the excited state(s). Conformational exchange rate constants (kex) of the TSA complex are all approximately 2500s-1, higher than any observed in the substrate-free enzyme (800-1900s-1). Elevated µs-ms timescale protein dynamics in the TSA-bound enzyme is more consistent with recently postulated catalytic networks involving multiple interconnected states at each step of the reaction, rather than a classical single stabilized transition state.

The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.

Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of µs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.

The modulation of haemolymph arginine kinase on the extracellular ATP induced bactericidal immune responses in the Pacific oyster Crassostrea gigas.

Arginine kinase is an important phosphagen kinase (PK) which plays an essential role in ATP buffering systems in invertebrates. In the present study, an arginine kinase (designated CgAK) was isolated by the lipopolysaccharide (LPS) affinity chromatography from the haemolymph of Crassostrea gigas. CgAK could directly bind to LPS in a concentration-dependent manner with the dissociation constant (Kd) of 2.46 × 10(-6) M. The interaction with LPS significantly decreased the ATP hydrolytic activity of CgAK, which in turn lead to the accumulation of ATP in vitro. The extracellular ATP stimulation could induce Ca(2+) influx, reactive oxygen species (ROS) production, and the release of lysosomal enzyme in the cellular immune response. In addition, ATP stimulation provoked the bactericidal activity towards Escherichia coli, and the scavenging ROS with N-acetyl-l-cysteine (NAC) abrogated the bactericidal activity, indicating that ATP stimulation could induce ROS-dependent antimicrobial activity in haemocytes. Collectively, the results demonstrated that the haemolymph CgAK could serve as an important purinergic regulator to modulate extracellular ATP, which might further have an important effect on the purinergic signaling-activated innate immune response of oyster.

Absolute Minimal Sampling in High-Dimensional NMR Spectroscopy.

Standard three-dimensional Fourier transform (FT) NMR experiments of molecular systems often involve prolonged measurement times due to extensive sampling required along the indirect time domains to obtain adequate spectral resolution. In recent years, a wealth of alternative sampling methods has been proposed to ease this bottleneck. However, due to their algorithmic complexity, for a given sample and experiment it is often hard to determine the minimal sampling requirement, and hence the maximal achievable experimental speed up. Herein we introduce an absolute minimal sampling (AMS) method that can be applied to common 3D NMR experiments. We show for the proteins ubiquitin and arginine kinase that for widely used experiments, such as 3D HNCO, accurate carbon frequencies can be obtained with a single time increment, while for others, such as 3D HN(CA)CO, all relevant information is obtained with as few as 6 increments amounting to a speed up of a factor 7-50.

Effects of high voltage pulsed electric field on structural properties and immune reactivity of arginine kinase in Fenneropenaeus chinensis.

To evaluate the effect of high voltage pulsed electric field (PEF) treatment (10-20 kV/cm, 5-15 min) on the structural characteristics and sensitization of crude extracts of arginine kinase from Fenneropenaeus chinensis. By simulated in vitro gastric juice digestion (SGF), intestinal juice digestion (SIF) and enzyme-linked immunosorbent assay (ELISA), AK sensitization was reduced by 42.5% when treated for 10 min at an electric field intensity of 15 kV/cm. After PEF treatment, the α-helix content decreased, and the α-helix content gradually changed to ß-sheet and ß-turn. Compared to the untreated group, the surface hydrophobicity increased and the sulfhydryl content decreased. SEM and AFM analyses showed that the treated sample surface formed a dense porous structure and increased roughness. The protein content, dielectric properties, and amino acid content of sample also changed significantly with the changes in the treatment conditions. Non-thermal PEF has potential applications in the development of hypoallergenic foods.

Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Comprehensive proteomics approach in characterizing and quantifying allergenic proteins from northern shrimp: toward better occupational asthma prevention.

Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m(3) for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens.

Crystal structure of shrimp arginine kinase in binary complex with arginine-a molecular view of the phosphagen precursor binding to the enzyme.

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta.

Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080 bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: K(m) arginine was 0.35 mM, K(m) ATP was 0.8-0.9 mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of the worm.

Identification of physicochemical properties of Scylla paramamosain allergen, arginin kinase.

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.

Identification and characterization of a putative arginine kinase homolog from Myxococcus xanthus required for fruiting body formation and cell differentiation.

Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.

Crystal structures of arginine kinase in complex with ADP, nitrate, and various phosphagen analogs.

Arginine kinase catalyzes the reversible transfer of a phosphoryl group between ATP and l-arginine and is a monomeric homolog of the human enzyme creatine kinase. Arginine and creatine kinases belongs to the phosphagen kinase family of enzymes, which consists of eight known members, each of which is specific for its own phosphagen. Here, the source of phosphagen specificity in arginine kinase is investigated through the use of phosphagen analogs. Crystal structures have been determined for Limulus polyphemus arginine kinase with one of four arginine analogs bound in a transition state analog complex: l-ornithine, l-citrulline, imino-l-ornithine, and d-arginine. In all complexes, the enzyme achieves a closed conformation very similar to that of the cognate transition state analog complex, but differences are observed in the configurations of bound ligands. Arginine kinase exhibits no detectable activity towards ornithine, citrulline, or imino-l-ornithine, and only trace activity towards d-arginine. The crystal structures presented here demonstrate that phosphagen specificity is derived neither from a lock-and-key mechanism nor a modulation of induced-fit conformational changes, but potentially from subtle distortions in bound substrate configurations.

Cloning and expression of arginine kinase from a swimming crab, Portunus trituberculatus.

Arginine kinase (AK) is an important phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the cDNA of AK (designated as PtAK) was identified from the eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA was 1,479 bp, containing an open reading frame of 1,074 bp that coded for 357 amino acids. The estimated molecular mass of mature PtAK was 40.30 kDa and theoretical isoelectric point was 6.18. Amino acid sequence alignment showed that PtAK had very high similarity with other shrimp and crab AKs ranging from 0.876 to 0.983. The genomic DNA fragments of about 1,434 bp consisted of two exons interrupted by an intron. Totally 24 SNPs, including 17 in the coding region and seven in the non-coding region, were detected by direct sequencing of 19 genomic samples. In exon 1, the coding SNPs (cSNPs) were only found in the disease-resistant specimens. The fluorescent real-time PCR analysis revealed that the expression of PtAK was detected in all the examined tissues with the highest expression in the muscle and the lowest in the eyestalk. The expression of PtAK after Vibrio alginolyticus injection was tested in haemocytes, showing that two peak values were 5.01-fold (at 3 h) and 3.60-fold (at 24 h) compared with the control values, respectively. The results suggested that AK might play an important role in the immune response in crabs.

Crystallization and X-ray diffraction studies of arginine kinase from the white Pacific shrimp Litopenaeus vannamei.

Crystals of an unligated monomeric arginine kinase from the Pacific whiteleg shrimp Litopenaeus vannamei (LvAK) were successfully obtained using the microbatch method. Crystallization conditions and preliminary X-ray diffraction analysis to 1.25 Šresolution are reported. Data were collected at 100 K on NSLS beamline X6A. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.5, b = 70.2, c = 81.7 Å. One monomer per asymmetric unit was found, with a Matthews coefficient (V(M)) of 2.05 Å(3) Da(-1) and 40% solvent content. Initial phases were determined by molecular replacement using a homology model of LvAK as the search model. Refinement was performed with PHENIX, with final R(work) and R(free) values of 0.15 and 0.19, respectively. Biological analysis of the structure is currently in progress.

Induction of mud crab (Scylla paramamosain) tropomyosin and arginine kinase specific hypersensitivity in BALB/c mice.

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. The allergens tropomyosin (TM) and arginine kinase (AK) from mud crab (Scylla paramamosain) were purified to homogeneity. BALB/c female mice were sensitized with TM and AK by intragastric administration. Mice treated with normal saline served as the negative control (NC) group. RESULTS: Compared with NC group, mice that were treated with TM and AK developed reduced activity; meanwhile, their scratching behavior and specific-IgE level were increased. Specific-CD4 + T cells were significantly elevated in the splenocyte cultures of the mice upon TM and AK stimulation. However, compared with the positive control group (ovalbumin, OVA), there was no significant difference. The expression of IL-4 in culture cells stimulated by TM, AK, and OVA group showed significant differences from the NC group, respectively. CONCLUSION: These results indicated that a BALB/c mouse model for sensitization to TM and AK from mud crab was successfully established, and the Th2 response was observed, displaying increased immunoglobulin E levels, together with the production of interleukin 4 and allergic symptoms.