Sarcoplasmic Calcium-binding Protein Research Articles

Transcriptional changes of genes encoding sarcoplasmic reticulum calcium binding and up-taking proteins in normal and Duchenne muscular dystrophy dogs

BackgroundCytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins.MethodsGiven the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle.ResultsIn affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs.ConclusionsOur study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines.

Specific detection of crustacean allergens in food: Development of indirect competitive and sandwich ELISA targeting sarcoplasmic calcium binding protein

To overcome the problem of cross-reactivity between crustaceans, mollusks, insects, and mites in current crustacean allergen immunodetection, the significant stable crustacean allergen, sarcoplasmic calcium binding protein (SCP) was innovatively selected as the target of ELISA detection. An indirect competitive ELISA (icELISA) and a sandwich ELISA (sELISA) were constructed and evaluated with spiked model foods. The sELISA (LOD 0.001 mg/kg and LOQ 0.003 mg/kg) is more sensitive than icELISA (LOD 0.11 mg/kg and LOQ 0.56 mg/kg). But the icELISA presented a wider standard range (10–10000 ng/mL) and greater recoveries of SCP in matrices (84.04%–118.94%). Sufficient precision (repeatability and reproducibility <15%), excellent specificity distinguishing crustaceans from others, and adequate detectability in diverse commercial processed foods were observed from the SCP-based icELISA. These results demonstrated the feasibility and applicability of SCP-targeted icELISA and provided a new direction to improve the capacity for crustacean allergen detection.

Ecotoxicological impact of heavy metals on wild mud crabs (Scylla olivacea) in Malaysia: An integrative approach of omics, molecular docking and human risk assessment

Mud crab, one of the aquatic organisms found in estuary areas, has become a significant economic source of seafood for communities due to its delectable taste. However, they face the threat of heavy metal contamination, which may adversely affect their biological traits. This study explored the comparison of the mud crabs collected from Setiu Wetland as a reference site, while Kuala Sepetang is an area that contains a higher concentration of heavy metals than Setiu Wetlands. Heavy metal levels were quantified using inductively coupled plasma mass spectrometry (ICP-MS), while proteomes were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 1H nuclear magnetic resonance (NMR)-based metabolomics, respectively. Heavy metal contamination affects the proteome, metabolome, and putative molecular targets in mud crabs (Scylla olivacea), leading to oxidative stress. Mud crabs collected from the metal-polluted area of Kuala Sepetang in Perak had considerably elevated concentrations of nickel (Ni), copper (Cu), zinc (Zn), lead (Pb), chromium (Cr), and cadmium (Cd) in comparison to the reference site of Setiu Wetlands in Terengganu. The proteome analysis revealed an upregulation of the stress-response protein Hsp70, which triggered superoxide dismutase (SOD) and increased arginine kinase expression (5.47 fold) in the muscle tissue, results in the alteration of metabolite regulation in the mud crab from Kuala Sepetang. Additionally, in the muscle tissues of mud crabs obtained from Kuala Sepetang, uncharacterized myosin-tail 1 domain proteins and sarcoplasmic calcium-binding proteins were downregulated. The metabolomic investigation identified changes in metabolites associated with energy metabolism and osmoregulation. Exploration of docking analysis suggests potential connections between methylarsonic acid and essential proteins in mud crabs. These findings suggest that the presence of heavy metals disrupts physiological processes and highlights potential molecular targets that warrant further investigation.

Gastropod Allergy: A Comprehensive Narrative Review.

Food allergies have increased significantly in recent decades, with shellfish being a leading cause of food allergy and anaphylaxis worldwide, affecting both children and adults. The prevalence of shellfish allergies is estimated to be approximately 0.5-2.5% of the general population, varying significantly by geographical location, age, and consumption habits. Although mollusk consumption has risen, the prevalence of mollusk allergies remains unknown. While extensive research has focused on crustacean allergies, mollusk allergies, particularly those related to gastropods, have received comparatively less attention. Clinical manifestations of shellfish allergy range from localized symptoms to life-threatening systemic reactions, such as anaphylaxis. Notably, severe bronchospasm is a predominant clinical feature in cases involving gastropods. Several allergens have been identified in mollusks, including paramyosin, tropomyosin, and sarcoplasmic calcium-binding protein. In gastropods, documented allergens include tropomyosin, paramyosin, the heavy chain of myosin, and Der p 4 amylase. Diagnosis typically involves a thorough clinical history, skin testing, in vitro quantification of immunoglobulin (Ig) E, and confirmation through an oral challenge, although the latter is reserved for selected cases. This narrative review highlights the limited research on gastropod allergy. It provides a comprehensive list of purified and recombinant allergens and discusses the applications of component-resolved diagnosis as well as current therapeutic developments.

Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics.

Sarcoplasmic calcium-binding protein (Cra a 4) from Crassostrea angulata belongs to the EF-hand superfamily, and understanding of its structure-allergenicity relationship is still insufficient. In this study, chemical denaturants were used to destroy the structure of Cra a 4, showing that disruption of the structure reduced its IgG-/IgE-binding activity. To explore which critical amino acid site affects the allergenicity of Cra a 4, the mutants were obtained by site-directed mutations in the disulfide bonds site (C97), conformational epitopes (I105, D114), or Ca2+-binding region (D106, D110) and their IgG-/IgE-binding activity was reduced significantly using serological tests. Notably, C97A had the lowest immunoreactivity. In addition, two conformational epitopes of Cra 4 were verified. Meanwhile, the increase of the α-helical content, surface hydrophobicity, and surface electrostatic potential of C97A affected its allergenicity. Overall, the understanding of the structure-allergenicity relationship of Cra a 4 allowed the development of a hypoallergenic mutant.

Allergen Diversity and Abundance in Different Tissues of the Redclaw Crayfish (Cherax quadricarinatus).

Shellfish allergy affects ~2.5% of the global population and is a type I immune response resulting from exposure to crustacean and/or molluscan proteins. The Australian Redclaw crayfish (Cherax quadricarinatus) is a freshwater species endemic to and farmed in northern Australia and is becoming an aquaculture species of interest globally. Despite being consumed as food, allergenic proteins from redclaw have not been identified or characterised. In addition, as different body parts are often consumed, it is conceivable that redclaw tissues vary in allergenicity depending on tissue type and function. To better understand food-derived allergenicity, this study characterised allergenic proteins in various redclaw body tissues (the tail, claw, and cephalothorax) and how the stability of allergenic proteins was affected through cooking (raw vs. cooked tissues). The potential of redclaw allergens to cross-react and cause IgE-binding in patients allergic to other shellfish (i.e., shrimp) was also investigated. Raw and cooked extracts were prepared from each body part. SDS-PAGE followed by immunoblotting was performed to determine allergen-specific antibody reactivity to sarcoplasmic calcium-binding protein and hemocyanin, as well as to identify redclaw proteins binding to IgE antibodies from individual and pooled sera of shrimp-allergic patients. Liquid chromatography-mass spectrometry (LC/MS) was utilised to identify proteins and to determine the proportion within extracts. Known crustacean allergens were found in all tissues, with a variation in tissue distribution (e.g., higher levels of hemocyanin in the claw and cephalothorax than in the tail). The proportion of some allergens as a percentage of remaining heat-stable proteins increased in cooked tissues. Previously described heat-stable allergens (i.e., hemocyanin and sarcoplasmic calcium-binding protein) were found to be partially heat-labile. Immunoblotting indicated that shrimp-allergic patients cross-react to redclaw allergens. IgE-binding bands, analysed by LC/MS, identified up to 11 known shellfish allergens. The findings of this study provide fundamental knowledge into the diagnostic and therapeutic field of shellfish allergy.

Development of a fluorescent multiplexed lateral flow immunoassay for the simultaneous detection of crustacean allergen tropomyosin, sarcoplasmic calcium binding protein and egg allergen ovalbumin in different matrices and commercial foods

A quantum dot (QD) based multiplexed lateral flow immunoassay (xLFIA) for the simultaneous detection of egg allergen ovalbumin, crustacean allergen tropomyosin (TM) and sarcoplasmic calcium binding protein (SCP) was developed in this study. QD-labeled rabbit anti-ovalbumin, SCP and TM antibodies were applied as fluorescent detection probes. The chromatography system was optimized to reduce the mutual interference of different test lines. Visual and instrumental detection limits of the xLFIA were 0.1 and 0.05 μg/mL for SCP, both 0.05 μg/mL for ovalbumin and both 0.5 μg/mL for TM. As low as 0.10 % crab powder, 0.01 % egg white powder and 0.05 % shrimp powder could be detected in all three model foods using xLFIA. Besides, the xLFIA detection results of 23 of 28 commercial foods were consistent with ingredient labels. These findings indicate that the developed xLFIA is a practical tool for point-of-care detection of egg and crustacean allergens in processed and commercial foods.

Purification, Expression, and Characterization of Sarcoplasmic Calcium Binding Protein: A Novel Allergen of Portunus trituberculatus.

Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgE-binding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4-70 °C and pH 3-10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.

IgE-Mediated Shellfish Allergy in Children.

Shellfish, including various species of mollusks (e.g., mussels, clams, and oysters) and crustaceans (e.g., shrimp, prawn, lobster, and crab), have been a keystone of healthy dietary recommendations due to their valuable protein content. In parallel with their consumption, allergic reactions related to shellfish may be increasing. Adverse reactions to shellfish are classified into different groups: (1) Immunological reactions, including IgE and non-IgE allergic reactions; (2) non-immunological reactions, including toxic reactions and food intolerance. The IgE-mediated reactions occur within about two hours after ingestion of the shellfish and range from urticaria, angioedema, nausea, and vomiting to respiratory signs and symptoms such as bronchospasm, laryngeal oedema, and anaphylaxis. The most common allergenic proteins involved in IgE-mediated allergic reactions to shellfish include tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, troponin c, and triosephosphate isomerase. Over the past decades, the knowledge gained on the identification of the molecular features of different shellfish allergens improved the diagnosis and the potential design of allergen immunotherapy for shellfish allergy. Unfortunately, immunotherapeutic studies and some diagnostic tools are still restricted in a research context and need to be validated before being implemented into clinical practice. However, they seem promising for improving management strategies for shellfish allergy. In this review, epidemiology, pathogenesis, clinical features, diagnosis, and management of shellfish allergies in children are presented. The cross-reactivity among different forms of shellfish and immunotherapeutic approaches, including unmodified allergens, hypoallergens, peptide-based, and DNA-based vaccines, are also addressed.

Immunological comparison of recombinant shrimp allergen Pen m 4, produced in Pichia pastoris and Escherichia coli.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3 to 2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.

Reduced Allergenicity of Shrimp (Penaeus vannamei) by Altering the Protein Fold, Digestion Susceptibility, and Allergen Epitopes.

The mechanism by which thermal/pressure processing influences the allergenicity of shrimp (Penaeus vannamei) was explored by anaphylaxis in mice, the protein structure, gastrointestinal digestion, and linear epitopes. Roasting induced the unfolding of the structure, which may reduce the allergenicity, but it made more linear epitopes to be exposed, causing mice to exhibit similar systemic anaphylaxis as mice fed with the raw shrimp protein (p > 0.05). However, the roasted + reverse-pressure-sterilized shrimp can significantly reduce specific antibodies, mast cell degranulation, vascular permeability, and histopathological morphology in mice compared with the raw and roasted shrimp (p < 0.05) because reverse-pressure sterilization causes protein to aggregate, hiding the heat/digested stable epitopes of arginine kinase (Glu59-Ser63, Asn112-Lys118, Leu131-Phe136, and Ser158-Glu162) and sarcoplasmic calcium-binding protein (Asn57-Phe67, Ser159-Cys165, and Glu126-Ala130) inside a 3D structure, while gastrointestinal digestion can destroy immunodominant, minor epitopes and the epitopes exposed by roasting. Meanwhile, the low binding frequency of IgE to troponin C was also responsible for maintaining the hypoallergenicity of shrimp.

Crystal Structure Analysis of Sarcoplasmic-Calcium-Binding Protein: An Allergen in Scylla paramamosain.

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp70 and Asp18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.

Shrimp pmo-miR-750 regulates the expression of sarcoplasmic calcium-binding protein facilitating virus infection in Penaeus monodon

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and play crucial roles in antiviral responses. Penaeus monodon miR-750 (pmo-miR-750) was found to be strongly up-regulated in the late phase of white spot syndrome virus (WSSV) infection, but its function remains uncharacterized. Herein, the targets that were translationally down-regulated in the shrimp stomach following a pmo-miR-750 mimic injection were identified using two-dimensional gel electrophoresis. Sarcoplasmic calcium-binding protein (Scp) and actin1 (Act1) were revealed to be down-regulated protein spots. The genuine binding of pmo-miR-750 mimic to Scp but not Act1 mRNA was validated in vitro. In addition, a negative correlation between the Scp transcript and pmo-miR-750 expression level in WSSV-infected P. monodon stomach implies that pmo-miR-750 regulates Scp expression in vivo. When injected into WSSV-infected shrimp, the pmo-miR-750 mimic suppressed Scp expression but significantly increased the WSSV copy number. Consistent with the miRNA mimic-mediated Scp suppression, the loss of function assay of Scp in WSSV-challenged shrimp by RNA interference revealed a decreased survival rate with a dramatic increase in viral copy number. Besides that, apoptosis was activated in the hemocytes of the Scp knockdown shrimp upon WSSV infection. Collectively, our findings reveal that up-regulated pmo-miR-750 suppresses Scp expression at both the transcript and protein levels in the late stage of WSSV infection, which contributes to modulating apoptosis and eventually enabling viral propagation.

Insight into the mechanism of allergenicity decreasing in recombinant sarcoplasmic calcium-binding protein from shrimp (Litopenaeus vannamei) with thermal processing via spectroscopy and molecular dynamics simulation techniques

In the present study, sarcoplasmic calcium-binding protein (SCP) was first expressed in E. coli BL21 (DE3), and then identified based on immunoblotting and SCP amino acid sequencing of shrimp (Litopenaeus vannamei) using mass spectrometry (MS). The recombinant SCP (rSCP) was treated with different temperature conditions to investigate its immunological properties, in vitro digestibility and structural changes with enzyme-linked immunosorbent assay (ELISA), immunoblotting, spectrophotometry and molecular dynamics simulation techniques. The immunoglobulin (Ig) E-binding activity of the rSCP could remain stable until 80 °C, whereas the higher thermal processing temperatures resulted in a significant decrease in IgG/IgE-binding capacity coupled with alterations in the secondary and tertiary structures. Notably, the maximum reduction of IgG/IgE reactivity and in vitro digestibility were observed in the autoclaved rSCP. The decrease in the potential allergenicity of rSCP not only correlated well with the decreasing of α-helix, epitopes masking and exposure of more protease cleavage sites, but also with the destruction of Ca2+ binding sites due to the unfolding of the rSCP with heating treatments, which was supported by the thermal-induced changes of the secondary and tertiary structures. These findings indicate that autoclaved treatment may be an effective and promising approach for producing hypoallergenic seafood.

Comprehending the allergen repertoire of shrimp for precision molecular diagnosis of shrimp allergy.

Clinical management of shrimp allergy is hampered by the lack of accurate tests. Molecular diagnosis has been shown to more accurately reflect the clinical reactivity but the full spectrum of shrimp allergens and their clinical relevance are yet to be established. We therefore sought to comprehend the allergen repertoire of shrimp, investigate and compare the sensitization pattern and diagnostic value of the allergens in allergic subjects of two distinct populations. Sera were collected from 85 subjects with challenge-proven or doctor-diagnosed shrimp allergy in Hong Kong and Thailand. The IgE-binding proteins of Penaeus monodon were probed by Western blotting and identified by mass spectrometry. Recombinant shrimp allergens were synthesized and analyzed for IgE sensitization by ELISA. Ten IgE-binding proteins were identified, and a comprehensive panel of 11 recombinant shrimp allergens was generated. The major shrimp allergens among Hong Kong subjects were troponin C (Pen m 6) and glycogen phosphorylase (Pen m 14, 47.1%), tropomyosin (Pen m 1, 41.2%) and sarcoplasmic-calcium binding protein (Pen m 4, 35.3%), while those among Thai subjects were Pen m 1 (68.8%), Pen m 6 (50.0%) and fatty acid-binding protein (Pen m 13, 37.5%). Component-based tests yielded significantly higher area under curve values (0.77-0.96) than shrimp extract-IgE test (0.70-0.75). Yet the best component test differed between populations; Pen m 1-IgE test added diagnostic value only in the Thai cohort, whereas sensitizations to other components were better predictors of shrimp allergy in Hong Kong patients. Pen m 14 was identified as a novel shrimp allergen predictive of challenge outcome. Molecular diagnosis better predicts shrimp allergy than conventional tests, but the relevant component is population dependent.

Major shrimp allergen peptidomics signatures and potential biomarkers of heat processing

It is important to develop tools that can be used to understand the effects of processing on allergenic foods in order to achieve personalized food labeling. To evaluate the effect of heating on the allergy-relevant structural properties of tropomyosin (TM), arginine kinase (AK), myosin light chain (MLC) and sarcoplasmic calcium-binding protein (SCP) shrimp allergens, trypsin digests of raw, fried and baked shrimp extracts were analyzed by peptidomics and epitope correlations. Processing altered the number of peptides released from the distinct allergens, and each treatment generated a specific epitope-matched peptide allergen fingerprint. Among the four allergens, TM led to a number of released peptides and epitope changes being detected, and AK provided the epitope-matched 331MGLTEFQAVK340 sequence as a common differentiating peptide for heat processing. These results provide new views on the structural effects of processing on major shrimp allergens and peptide candidates as processing biomarkers.

Insight into IgG/IgE binding ability, in vitro digestibility and structural changes of shrimp (Litopenaeus vannamei) soluble extracts with thermal processing

The effects of six kinds of thermal processing on soluble protein recovery, potential allergenicity, in vitro digestibility and structural characteristics of shrimp soluble proteins were evaluated. Obtained results confirmed soluble protein recovery and IgG/IgE reactivity of shrimp soluble extracts were markedly suppressed by various thermal treatments with enhanced digestibility depended on the extent and type of heating applied, which correlated well with the structural alterations and modification. The maximum reduction of IgG/IgE-binding capacity and digestive stability were observed in the autoclaved shrimps because of unfolding of protein and hydrophobic residues exposed. Notably, tropomyosin (TM) and sarcoplasmic calcium-binding protein (SCP) were still IgG/IgE-reactive in various heat-processed shrimps, even higher IgG reactivity were found in heat-treated shrimps TM according to TM antiserum western-blotting and indirect ELISA results. Shrimp TM and SCP maintains its IgE/IgG-binding capacity after various cooking methods, thus most probably initiating allergic sensitization to both raw and cooked shrimps.

Physicochemical characterization and identification of major linear epitopes of sarcoplasmic calcium-binding protein (SCP) allergen from Pacific oyster (Crassostrea gigas).

Food allergy is a serious public nutritional health problem that has attracted extensive worldwide attention. Shellfish allergy is a long-lasting disorder that has a lifelong impact on health. Sarcoplasmic calcium-binding protein (SCP) plays a vital role in cell and muscle functions and has been identified as an allergen in oyster. In this study, recombinant SCP (rSCP) with a molecular mass of 21 kDa was produced and identified based on SCP amino acid sequencing of Pacific oyster (Crassostrea gigas), and was used as a follow-up experimental material. Its physicochemical characterization showed that purified rSCP is highly stable to heat and acid-alkali and trypsin digestion but less resistant to pepsin digestion. We established an animal sensitization model and rSCP displayed stronger Immunoglobulin E (IgE)-binding activity with rat serum in the rSCP + cholera toxin (CT) group compared with the CT group and a control group. Five epitope peptides were identified as linear immunodominant epitopes by indirect competitive enzyme-linked immunosorbent assay (icELISA) for the first time. We also found that conformational epitopes may play a major role in the immunoreactivity of SCP. These results are significant for understanding hypersensitization of humans to oyster and offer available preventive measures and treatment programs in further research. © 2021 Society of Chemical Industry.

Linear Epitopes Play an Important Role in the Immunoglobulin G (IgG)/Immunoglobulin E (IgE)-Binding Capacity of Scy p 4.

Sarcoplasmic calcium-binding protein is a stable allergen in Scylla paramamosain and named Scy p 4. To explore the importance of linear epitopes in the immunoglobulin G (IgG)/immunoglobulin E (IgE)-binding capacity of Scy p 4, chemical denaturants were used to destroy the structure. Scy p 4 was reduced with dithiothreitol and subsequently alkylated with iodoacetamide (IAA). Furthermore, the structural analysis indicated that IAA-Scy p 4 was an unstructured protein. The inhibition enzyme-linked immunosorbent assay showed that IAA-Scy p 4 could inhibit the binding of Scy p 4 to sensitize serum, with inhibition rates reached 55%. Moreover, the linear mimotopes of Scy p 4 were predicted in silico. Three linear epitopes were verified by serological tests and named L-Scy p 4-1 (AA76-91), L-Scy p 4-2 (AA111-125), and L-Scy p 4-3 (AA137-146). Overall, these data provide an understanding of the relationship between the structure and allergenicity about Scy p 4, and the identified linear epitopes can be used for diagnosis and food processing of shellfish allergy.