Cloning, Expression, and the Effects of Processing on Sarcoplasmic-Calcium-Binding Protein: An Important Allergen in Mud Crab.

Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.

Molecular and cellular analyses of a ryanodine receptor from hemocytes of Pieris rapae.

Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are a distinct class of ligand-gated calcium channels controlling the release of calcium from intracellular stores. Intracellular calcium level has a definite role in innate and adaptive immune signaling. However, very few information are accessible about calcium transients of invertebrate immunocytes, especially of insect hemocytes, the effector cells of insect immunity. In this study, we show that the RyR-stimulating agent flubendiamide inhibit hemocyte spreading and phagocytosis in the cabbage white butterfly, Pieris rapae. Furthermore, we cloned a cDNA encoding a ryanodine receptor (PrRyR) from the hemocytes of P. rapae. It encodes 5107 amino acids with a predicted molecular weight of 578.2 kDa. PrRyR shares a common feature with known RyRs: a well-conserved COOH-terminal domain with two consensus calcium-binding EF-hands and six transmembrane domains, and a large hydrophilic NH2-terminal domain. In the larval stage, PrRyR was highly expressed in epidermis tissue and also expressed in hemocytes at a moderate level. In the adult stage, PrRyR was expressed at high levels in thoraces and legs, while low levels in abdomens and antennae. Quantitative real-time PCR analysis showed that its expression did not display any significant change in response to bacterial challenge. Western blot analysis and immunohistochemistry assay displayed that PrRyR was detected and presented on hemocytes. We also showed that flubendiamide, a RyR-activating insecticide, induced Ca(2+) release and thereby confirmed functional expression of the PrRyR in the hemocytes of P. rapae.

Purification and characterisation of sarcoplasmic calcium-binding protein, a novel allergen of red swamp crayfish (Procambarus clarkii).

Crayfish sarcoplasmic calcium-binding protein (SCP) was purified. The physicochemical and polymorphic characterisations were also analysed. SCP was purified by column chromatography to reveal a single band with molecular mass of 22 kDa and further confirmed by mass spectrometry. The results of physicochemical characterisation showed that SCP was stable in the processes of thermal or acid/alkali treatment, and could be digested by simulate gastrointestinal fluid. Importantly, the comparison of SCP polymorphism using sera from crustacean-allergic patients demonstrated SCP-II had a weaker IgE-binding activity. The isoelectric points of SCP subunits a, b and c were 4.6, 4.7, and 4.8, respectively, as determined by two-dimensional electrophoresis and IgE immunoblotting analysis showed that patients' sera reacted to three subunits of SCP. Finally, it can be concluded that SCP is a stable polymorphic allergen in crayfish, and all of its isotypes and subunits have allergenicity.

[Mass spectrometry identification and immune cross-reactivity of a minor shrimp allergen-sarcoplasmic calcium binding protein from Litopenaeus vannamei].

AIM: To identify sarcoplasmic calcium-binding protein (SCP) as a minor shrimp allergen by mass spectrometry, and to analyze the immune cross-reactivity among crustacean SCPs. METHODS: The M(r); 21 000 allergen from Litopenaeus vannamei was identified by MALDI-TOF/TOF-MS. BLAST and ClustalW were used to compare amino acid sequence identity of the allergen among crustaceans. The puritifed M(r); 21 000 allergen was injected subcutaneously in mice to produce the specific polyclonal antibodies to analyze immune cross-reactivity of the allergen with proteins from 8 other species of crustaceans by Western blotting. RESULTS: The M(r); 21 000 shrimp allergen was identified as SCP. Sequence comparison revealed that SCP had 81%-100% amino acid identity among crustaceans. Western blotting showed that the proteins with M(r); about 21 000, corresponding to SCP from Metapenaeus ensis, Penaeus monodon, Oratosquilla oratoria, Macrobrachium rosenbergii, Procambarus clarkii, Portunus pelagicus, Charybdis feriatus, Eriocheir sinensis were recognized by polyclonal antibodies against SCP of Litopenaeus vannamei. CONCLUSION: SCP is a minor shrimp allergen, and SCPs have a high sequence homology and strong immune cross-reactivity among crustaceans, which can be used as detective, diagnostic and safe immunotherapeutic agents for subjects with shrimp allergy.

Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells.

A number of immunological functions are ascribed to cell surface-expressed forms of the endoplasmic reticulum (ER) chaperone calreticulin (CRT). In this study, we examined the impact of ER stress-inducing drugs upon cell surface CRT induction and the resulting immunological consequences. We showed that cell surface expression of CRT and secretion of CRT, BiP, gp96, and PDI were induced by thapsigargin (THP) treatment, which depletes ER calcium, but not by tunicamycin treatment, which inhibits protein glycosylation. Surface expression of CRT in viable, THP-treated fibroblasts correlated with their enhanced phagocytic uptake by bone marrow-derived dendritic cells. Incubation of bone marrow-derived dendritic cells with THP-treated fibroblasts enhanced sterile IL-6 production and LPS-induced generation of IL-1ß, IL-12, IL-23, and TNF-α. However, extracellular CRT is not required for enhanced proinflammatory responses. Furthermore, the pattern of proinflammatory cytokine induction by THP-treated cells and cell supernatants resembled that induced by THP itself and indicated that other ER chaperones present in supernatants of THP-treated cells also do not contribute to induction of the innate immune response. Thus, secretion of various ER chaperones, including CRT, is induced by ER calcium depletion. CRT, previously suggested as an eat-me signal in dead and dying cellular contexts, can also promote phagocytic uptake of cells subject to ER calcium depletion. Finally, there is a strong synergy between calcium depletion in the ER and sterile IL-6, as well as LPS-dependent IL-1ß, IL-12, IL-23, and TNF-α innate responses, findings that have implications for understanding inflammatory diseases that originate in the ER.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

BACKGROUND: Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. METHODS: A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. RESULTS: The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. CONCLUSIONS: SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish.

[Transfer factor of immune reactivity to diphtheria-tetanus anatoxin modulates the action of neurotransmitters in the intestinal smooth muscle].

Transfer factor (TF) of immune reactivity (10(-5) - 10(-3) mg/ml) to diphtheria-tetanus anatoxin modulates slow waves and spontaneous contractile activity of non-atropinized smooth muscle stripes (SMS) of guinea-pig taenia coli. TF (10(-4) mg/ml) transforms slow waves into stable depolarization and tonic contraction. After SMS atropinization, the substance acts in the same way. In the presence of methylene blue (10(-5) M), a guanylatecyclase blocker, FT induces transitory increase of SMS muscle tone, which is followed by their stable relaxation. ATP and UTP, purinoceptors agonists, evoke substantial hyperpolarization of smooth muscle cells membrane and their relaxation. FT enhances post-inhibitory excitation in SMS. In the presence of acetylcholine (10(-5) M) FT (10(-4) mg/ml) transforms the inhibitory ATP action on tonic contraction into excitative. This substance (10(-5), 10(-4) mg/ml) enhances Ca2+ mobilization from ryanodine-sensitive calcium store, inhibits the release of these cations from IP3-sensitive calcium store of sarcoplasmic reticulum. TF demolishes the inhibitory actions of sodium nitroprusside (nitric oxide donor), and noradrenaline in taenia coli smooth muscles.

[The effect of antibodies on the Ca2+ release from the sarcoplasmic reticulum of the papillary muscle of the rat heart]. / Vplyv antytil na vykhid Ca2+ iz sarkoplazmatychnoho retikuluma papiliarnoho m'iaza sertsia shchura.

In experiments of skinned with saponin papillary muscles (PM) of the rat's heart we studied the effects of antibodies (AB), which are specific for the sarcoplasmic reticulum's membranes (SPR). AB (1-5 mg protein/ml) have been established to enhance the amplitude of the isometric PM tension. Inhibition of the Ca2+ release from SPR and its previous release prevented the development of the exciting effect of AB. Thus, AB increase the amplitude of PM contraction due to their intracellular mobilizing action.

Myasthenia gravis sera containing antiryanodine receptor antibodies inhibit binding of [3H]-ryanodine to sarcoplasmic reticulum.

Myasthenia gravis (MG) patients with thymoma often have antibodies against the calcium-release channel of the sarcoplasmic reticulum (SR) in striated muscle, the ryanodine receptor (RyR). RyR function can be tested in vitro by measuring the degree of [3H]-ryanodine binding to SR. In this study, sera from 9 out of 14 MG patients containing RyR antibodies inhibited [3H]-ryanodine binding to SR membranes from rat skeletal muscle. The 9 patients with antibodies inhibiting ryanodine binding had more severe MG than those with noninhibiting antibodies (P = 0.006). Sera from MG patients with acetylcholine receptor and titin muscle antibodies but no antibodies against RyR and blood-donor sera did not have an inhibiting effect in the [3H]-ryanodine binding assay. The results show that RyR antibodies in MG patients have high affinity for the RyR, and that the binding of antibodies probably affects calcium release from SR by locking the RyR ion channel in a closed position.

Human leukocyte antigen class I in polymyositis: leukocyte infiltrates, regeneration, and impulse block.

In polymyositis (PM), T-cell mediated myocytotoxicity is directed against strongly human leukocyte antigen class I positive (HLA-I+) muscle fibers. Fiber regeneration probably is partly responsible for this HLA-I up-regulation. We have evaluated regeneration, denervation/impulse blockade, and focal leukocyte infiltrates as possible HLA-I inducing factors in PM. Distinctive patterns of HLA-I, nerve cell adhesion molecule (NCAM), and vimentin expression accompany denervation and regeneration. Regenerating fibers also have centralized nuclei. Using semiquantitative methods, we examined strongly HLA-I+ fibers in PM muscle biopsies for these markers. Sarcoplasmic HLA-I levels were related to the presence of leukocyte infiltrates and invasion of fibers. Strongly HLA-I+ fibers were frequently invaded, and regeneration-associated changes were usually observed at sites of fiber damage. Sarcoplasmic HLA-I levels were stable along intact fibers, also adjacent to leukocyte infiltrates. A majority of the strongly HLA-I+ fibers were nonregenerating (NCAM+ only). Though other mechanisms cannot be excluded, this suggests that impulse blockade or denervation may contribute to extra HLA-I up-regulation in these fibers.

Modulation of endoplasmic reticulum calcium pump expression during T lymphocyte activation.

Calcium mobilization from intracellular storage organelles is a key component of the second messenger system inducing cell activation. Calcium transport ATPases associated with intracellular calcium storage organelles play a major role in controlling this process by accumulating calcium from the cytosol into intracellular calcium pools. In this study the modulation of the expression of the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) isoenzymes has been studied in lymphocytes undergoing phorbol myristate acetate and ionomycin-induced activation. In several T lymphocyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the expression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expression of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied separately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concomitantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activation induces a selective and cyclosporine-A-sensitive modulation of the expression of the SERCA calcium pump isoforms. This reflects a profound reorganization of the calcium homeostasis of T cells undergoing activation and may open new avenues in the understanding of the plasticity of the calcium homeostasis of differentiating cells and in the pharmacological modulation of lymphocyte function.

SR-Ca2+ ATPase as an autoimmunogen in experimental myocarditis.

The concept of autoimmunity in the pathogenesis of myocarditis or dilated cardiomyopathy is gaining impetus. Since systolic functional impairment and subsequent recovery are frequently observed in myocarditis, we reasoned that the development of autoimmunity to cardiac sarcoplasmic reticulum calcium ATPase (SR-Ca2+ ATPase), which could interfere with intracellular calcium regulation and therefore affect myocardial contractility, should lead to immune-mediated myocarditis in experimental animals. Murine monoclonal antibody 4C11-20.21 (IgM class) generated against canine cardiac SR-Ca2+ ATPase inhibits the cardiac but not the skeletal ATPase activity. Immunization of CAF1/J mice with 4C11-20.21-affinity-column-purified cardiac SR-ATPase produced a time-dependent induction of myocardial injury consistent with the diagnosis of myocarditis. Furthermore, the antibody 4C11-20.21 alone can induce myo-necrosis in severe combined immunodeficiency (SCID) mice indicating a mechanism of cardiomyopathy independent of the cytotoxic T-cell mediated autoimmunopathy. Administration of 4C11-20.21 into immunocompetent CAF1/J mice resulted in minimal myocardial abnormality (40% with perivascular and/or interstitial mononuclear lymphoplasmacytoid aggregates, 10% with borderline myocarditis and 10% with lesions consistent with focal myocarditis). All control animals had normal hearts. Immunoperoxidase electron microscopic examination of the involved cardiac tissues showed antibody localization in the subsarcolemmal myotubular system and focal staining of the immediately adjacent sarcolemma in mice injected with 4C11-20.21 but not with 2C12.1B5. The time-dependent association between cardiac SR-Ca2+ ATPase administration and development of myocardial lesions, as well as potentiated induction of myonecrosis with anti-cardiac SR-Ca2+ ATPase antibody in SCID relative to immunocompetent mice, suggest a potential autoimmunopathogenic role of cardiac SR-Ca2+ ATPase in experimental myocarditis.

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle.

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T-tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.

Identification of heterotrimeric and low molecular weight GTP-binding proteins in rabbit skeletal muscle longitudinal sarcoplasmic reticulum.

Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of Gs alpha, showed an immunoreactive band at about 45 kDa. [32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)-ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a Gs alpha, the absence of Gi and G(o), and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.

Ryanodine receptor autoantibodies in myasthenia gravis patients with a thymoma.

Sera from patients with myasthenia gravis were examined by Western blot for the presence of antibodies to proteins of the sarcoplasmic reticulum from rabbit skeletal muscle. Fourteen of 30 patients with myasthenia gravis and a thymoma had IgG autoantibodies to the calcium release channel of the sarcoplasmic reticulum (the ryanodine receptor), which plays a crucial role in the mechanism of excitation-contraction coupling in striated muscle. Ryanodine receptor autoantibodies were not detected in any of the 45 sera from patients with myasthenia gravis without a thymoma. Ryanodine receptor autoantibodies may have pathogenetic relevance in thymoma-associated myasthenia gravis.

Immunological relatedness of the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase.

The effect of anti-ATPase antibodies with epitopes near Asp-351 (PR-8), Lys-515 (PR-11) and the ATP binding domain (D12) of the Ca(2+)-ATPase of sarcoplasmic reticulum (EC 3.6.1.38) was analyzed. The PR-8 and D12 antibodies reacted freely with the Ca(2+)-ATPase in the native membrane, indicating that their epitopes are exposed on the cytoplasmic surface. Both PR-8 and D12 interfered with the crystallization of the Ca(2+)-ATPase, suggesting that their binding sites are at interfaces between ATPase molecules. PR-11 had no effect on ATPase-ATPase interactions or on the ATPase activity of sarcoplasmic reticulum. The epitope of PR-11 is suggested to be the VIDRC sequence at residues 520-525, while that of D12 at residues 670-720 of the Ca(2+)-ATPase. The use of predictive algorithms of antigenicity for identification of potential antigenic determinants in the Ca(2+)-ATPase is analyzed.

Immune-mediated modulation of sarcoplasmic reticulum function in human dilated cardiomyopathy.

Calcium transport by the cardiac sarcoplasmic reticulum is depressed in human dilated cardiomyopathy, but the mechanisms involved are not clear. The possible involvement of immunological mechanisms was explored by evaluating the effect of sera from 49 patients with dilated cardiomyopathy on oxalate-facilitated Ca2+ uptake. In 14 of these patients, serum or IgG induced a time- and concentration-dependent decline (29 +/- 4% at 100-fold serum dilution) in Ca2+ transport. In 14 patients, autoantibodies against the beta 1-adrenoceptor were also demonstrated by a ligand binding inhibition assay. Serum from these patients inhibited the isoproterenol-mediated stimulation of Ca2+ uptake in permeabilized cardiac myocytes, but did not prevent the effect of protein kinase A. Anti-beta-receptor antibodies were present in 50% of the sera inhibiting Ca2+ uptake compared to 20% of those without inhibitory activity, (p less than 0.01). There was a strong correlation between the inhibition of sarcoplasmic reticulum Ca2+ transport and the HLA-DR4 phenotype (78% compared to 30% in patients with no inhibitory effect). These results suggest that immunological mechanisms play an important role in modifying sarcoplasmic reticulum function in about a third of the patients with detailed cardiomyopathy.