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• W2920252492 abstract "IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)–treated IgE cannot access its binding site on bone marrow–derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)–treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies. IgE plays a key role in allergies by binding to allergens and then sensitizing mast cells through the Fc receptor, resulting in the secretion of proinflammatory mediators. Therefore, IgE is a major target for managing allergies. Previous studies have reported that oligomannose on IgE can be a potential target to inhibit allergic responses. However, enzymes that can modulate IgE activity are not yet known. Here, we found that the commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid specifically modulates IgE, but not IgG, and prevents the initiation of anaphylaxis. RDE (II)–treated IgE cannot access its binding site on bone marrow–derived mast cells, resulting in reduced release of histamine and cytokines. We also noted that RDE (II)–treated IgE could not induce passive cutaneous anaphylaxis in mouse ears. Taken together, we concluded that RDE (II) modulates the IgE structure and renders it unable to mediate allergic responses. To reveal the mechanism by which RDE (II) interferes with IgE activity, we performed lectin microarray analysis to unravel the relationship between IgE modulation and glycosylation. We observed that RDE (II) treatment significantly reduced the binding of IgE to Lycopersicon esculentum lectin, which recognizes poly-N-acetylglucosamine and poly-N-acetyllactosamine. These results suggest that RDE (II) specifically modulates branched glycans on IgE, thereby interfering with its ability to induce allergic responses. Our findings may provide a basis for the development of drugs to inhibit IgE activity in allergies. Immunoglobulin ε (IgE) plays a key role in type I allergy (e.g. asthma and anaphylaxis). IgE was discovered about 50 years ago by Ishizaka and Ishizaka (1Ishizaka K. Ishizaka T. Identification of γ-E-antibodies as a carrier of reaginic activity.J. Immunol. 1967; 99 (4168663): 1187-1198PubMed Google Scholar, 2Ishizaka K. Ishizaka T. Identification of IgE.J. Allergy Clin. Immunol. 2016; 137 (27090936): 1646-165010.1016/j.jaci.2015.12.1343Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) as a novel immunoglobulin able to induce allergic reactions in the skin. The study found that intracutaneous injection with only 1–2 ng/ml IgE could induce an erythema-wheal reaction in healthy subjects (2Ishizaka K. Ishizaka T. Identification of IgE.J. Allergy Clin. Immunol. 2016; 137 (27090936): 1646-165010.1016/j.jaci.2015.12.1343Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mast cells and blood basophils in the tissue were found to express a high affinity to the IgE receptor, FcεRI 2The abbreviations used are: FcεRIhigh-affinity fragment crystallizable ε receptorBMMbone marrow–derived macrophageBMMCbone marrow–derived mast cellBNPPbis-p-nitrophenyl phosphate(light chain) BPbinding proteinCBBCoomassie Brilliant BlueDFPdiisopropyl fluorophosphateDNPdinitrophenolEndo F1endoglycosidase F1LacNAcN-acetyl-lactosamineRDEreceptor-destroying enzymeHAhemagglutininHSAhuman serum albuminHEK293Thuman embryonic kidney cells 293 that stably express the SV40 large T antigenLELL. esculentum lectinNAneuraminidase (sialidase)PCApassive cutaneous anaphylaxisPHA-LP. vulgaris leucoagglutininPNGase Fpeptide-N-glycosidase FTNPtrinitrophenylTNFαtumor necrosis factor-αHRPhorseradish peroxidasePVDFpolyvinylidene difluorideAPCallophycocyaninANOVAanalysis of varianceILinterleukin. (3Balbino B. Conde E. Marichal T. Starkl P. Reber L.L. Approaches to target IgE antibodies in allergic diseases.Pharmacol. Ther. 2018; 191 (29909239): 50-6410.1016/j.pharmthera.2018.05.015Crossref PubMed Scopus (32) Google Scholar, 4Cookson W. The immunogenetics of asthma and eczema: a new focus on the epithelium.Nat. Rev. Immunol. 2004; 4 (15573132): 978-98810.1038/nri1500Crossref PubMed Scopus (330) Google Scholar). By subsequent exposure to the allergen, IgE-binding mast cells released proinflammatory mediators, including histamine and cytokines, which cause an allergic response (5Yamaguchi M. Lantz C.S. Oettgen H.C. Katona I.M. Fleming T. Miyajima I. Kinet J.P. Galli S.J. IgE enhances mouse mast cell FcεRI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions.J. Exp. Med. 1997; 185 (9034145): 663-67210.1084/jem.185.4.663Crossref PubMed Scopus (390) Google Scholar). Yamaguchi et al. (5Yamaguchi M. Lantz C.S. Oettgen H.C. Katona I.M. Fleming T. Miyajima I. Kinet J.P. Galli S.J. IgE enhances mouse mast cell FcεRI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions.J. Exp. Med. 1997; 185 (9034145): 663-67210.1084/jem.185.4.663Crossref PubMed Scopus (390) Google Scholar) then demonstrated that IgE enhances the expression level of FcεRI in mast cells and permits mast cells to increase production of proinflammatory mediators by antigen challenge. Taken together, IgE is considered one of the major targets for therapy against allergies. Omalizumab, which binds to the Fc region of IgE and inhibits binding to FcεRI, has been previously found to be a successful therapy against certain allergies (3Balbino B. Conde E. Marichal T. Starkl P. Reber L.L. Approaches to target IgE antibodies in allergic diseases.Pharmacol. Ther. 2018; 191 (29909239): 50-6410.1016/j.pharmthera.2018.05.015Crossref PubMed Scopus (32) Google Scholar, 6Busse W. Corren J. Lanier B.Q. McAlary M. Fowler-Taylor A. Cioppa G.D. van As A. Gupta N. Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma.J. Allergy Clin. Immunol. 2001; 108 (11496232): 184-19010.1067/mai.2001.117880Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 7Oettgen H.C. Fifty years later: emerging functions of IgE antibodies in host defense, immune regulation, and allergic diseases.J. Allergy Clin. Immunol. 2016; 137 (27263999): 1631-164510.1016/j.jaci.2016.04.009Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, omalizumab cannot displace IgE bound to FcεRI, which leads to a delay of several weeks or months before the onset of any clinical benefits (7Oettgen H.C. Fifty years later: emerging functions of IgE antibodies in host defense, immune regulation, and allergic diseases.J. Allergy Clin. Immunol. 2016; 137 (27263999): 1631-164510.1016/j.jaci.2016.04.009Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). As such, another approach to IgE is necessary to develop a therapy against allergy. high-affinity fragment crystallizable ε receptor bone marrow–derived macrophage bone marrow–derived mast cell bis-p-nitrophenyl phosphate binding protein Coomassie Brilliant Blue diisopropyl fluorophosphate dinitrophenol endoglycosidase F1 N-acetyl-lactosamine receptor-destroying enzyme hemagglutinin human serum albumin human embryonic kidney cells 293 that stably express the SV40 large T antigen L. esculentum lectin neuraminidase (sialidase) passive cutaneous anaphylaxis P. vulgaris leucoagglutinin peptide-N-glycosidase F trinitrophenyl tumor necrosis factor-α horseradish peroxidase polyvinylidene difluoride allophycocyanin analysis of variance interleukin. Glycosylation of immunoglobulin is considered to be important for its structure and function (8Shade K.-T. Anthony R. Antibody glycosylation and inflammation.Antibodies. 2013; 2: 392-41410.3390/antib2030392Crossref Scopus (81) Google Scholar). Minor modifications of glycans on IgG (e.g. fucose depletion (9Natsume A. Niwa R. Satoh M. Improving effector functions of antibodies for cancer treatment: enhancing ADCC and CDC.Drug Des. Devel. Ther. 2009; 3 (19920917): 7-16PubMed Google Scholar)) can have a significant impact on receptor binding and the effector functions (8Shade K.-T. Anthony R. Antibody glycosylation and inflammation.Antibodies. 2013; 2: 392-41410.3390/antib2030392Crossref Scopus (81) Google Scholar). In contrast, IgE is the most heavily glycosylated antibody (10Shade K.-T. Platzer B. Washburn N. Mani V. Bartsch Y.C. Conroy M. Pagan J.D. Bosques C. Mempel T.R. Fiebiger E. Anthony R.M. A single glycan on IgE is indispensable for initiation of anaphylaxis.J. Exp. Med. 2015; 212 (25824821): 457-46710.1084/jem.20142182Crossref PubMed Scopus (83) Google Scholar, 11Wu G. Hitchen P.G. Panico M. North S.J. Barbouche M.R. Binet D. Morris H.R. Dell A. Haslam S.M. Glycoproteomic studies of IgE from a novel hyper-IgE syndrome linked to PGM3 mutation.Glycoconj. J. 2016; 33 (26687240): 447-45610.1007/s10719-015-9638-yCrossref PubMed Scopus (28) Google Scholar). Human IgE has seven predicted N-glycosylation sites, whereas murine IgE has eight or nine (8Shade K.-T. Anthony R. Antibody glycosylation and inflammation.Antibodies. 2013; 2: 392-41410.3390/antib2030392Crossref Scopus (81) Google Scholar, 10Shade K.-T. Platzer B. Washburn N. Mani V. Bartsch Y.C. Conroy M. Pagan J.D. Bosques C. Mempel T.R. Fiebiger E. Anthony R.M. A single glycan on IgE is indispensable for initiation of anaphylaxis.J. Exp. Med. 2015; 212 (25824821): 457-46710.1084/jem.20142182Crossref PubMed Scopus (83) Google Scholar). Björklund et al. (12Björklund J.E. Karlsson T. Magnusson C.G. N-Glycosylation influences epitope expression and receptor binding structures in human IgE.Mol. Immunol. 1999; 36 (10403487): 213-22110.1016/S0161-5890(99)00036-XCrossref PubMed Scopus (26) Google Scholar) reported that N-linked glycan is important for binding to FcεRI. They indicated that Flavobacterium meningosepticum peptide:N-glycosidase F (PNGase F), which removes almost all N-linked oligosaccharides, reduces the binding level of IgE to FcεRI in ELISA. Moreover, Shade et al. (10Shade K.-T. Platzer B. Washburn N. Mani V. Bartsch Y.C. Conroy M. Pagan J.D. Bosques C. Mempel T.R. Fiebiger E. Anthony R.M. A single glycan on IgE is indispensable for initiation of anaphylaxis.J. Exp. Med. 2015; 212 (25824821): 457-46710.1084/jem.20142182Crossref PubMed Scopus (83) Google Scholar) reported that oligomannose on Asn-394 in human IgE and Asn-384 in murine IgE is important for the structural integrity of the immunoglobulin. Modifications at these sites by endoglycosidase F1 (Endo F1), which cleaves within the chitobiose core of high-mannose and some hybrid oligosaccharides from N-linked glycoproteins, abrogate IgE binding to FcεRI. Therefore, the modification of oligomannose prevents the initiation of anaphylaxis by mast cells. In their study, they suggested that IgE oligomannose may be a potential therapeutic target. Wu et al. (11Wu G. Hitchen P.G. Panico M. North S.J. Barbouche M.R. Binet D. Morris H.R. Dell A. Haslam S.M. Glycoproteomic studies of IgE from a novel hyper-IgE syndrome linked to PGM3 mutation.Glycoconj. J. 2016; 33 (26687240): 447-45610.1007/s10719-015-9638-yCrossref PubMed Scopus (28) Google Scholar) also determined high-mannose glycans on the same site in IgE obtained from a patient with a novel hyper-IgE syndrome. However, PNGase F and Endo F1 cannot specifically modulate IgE because most sugar proteins have N-linked oligosaccharides. To target the glycan on IgE specifically for the therapy, more research is required to identify a specific enzyme that is able to modulate the glycan structure in IgE. Previously, we coincidentally found that commercial receptor-destroying enzyme (RDE) (II) from Vibrio cholerae culture fluid (13Tyrrell D.A. Horsfall Jr., F.L. A procedure which eliminates nonspecific inhibitor from human serum but does not affect specific antibodies against influenza viruses.J. Immunol. 1952; 69 (13011314): 563-574PubMed Google Scholar) reduced the binding level of IgE to influenza virus antigen, hemagglutinin (HA) (Fig. 1A) (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). RDE (II) contains a neuraminidase (NA) (sialidase) and is usually used for preventing nonspecific binding of influenza HA to sialic acid in neutralizing assays. We genetically switched the constant region of anti-HA IgG to anti-HA IgA, IgM, IgD, and IgE and established all classes of anti-HA–coding plasmids. All variable regions were the same, and the binding affinity to influenza HA was considered as equal. However, RDE (II)–treated IgE could not neutralize virus infection despite there being sufficient activity of RDE (II)–treated IgG, IgA, and IgM (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). In this study, we showed that RDE (II) altered the structure of IgE and abrogated IgE binding to FcεRI on mast cells, which is directly related to the mast cell degranulation, thereby preventing anaphylaxis. Furthermore, we identified that RDE (II)–treated IgE had reduced capacity of binding to Lycopersicon esculentum (LEL), which recognizes poly N-acetylglucosamine (GlcNAc) and poly-N-acetyllactosamine (LacNAc) (15Immel F. Broussard C. Catherinet B. Plasseraud L. Alcaraz G. Bundeleva I. Marin F. The shell of the invasive bivalve species Dreissena polymorpha: biochemical, elemental and textural investigations.PLoS ONE. 2016; 11 (27213644)e015426410.1371/journal.pone.0154264Crossref PubMed Scopus (25) Google Scholar16Ferreira-Medeiros M. Correa-Gillieron E. Recognition of N-acetylglucosamine (GlyNAc) and poly-N-acetyllactosamine residues in vessels of the rat pineal gland.Int. J. Morphol. 2004; 22: 285-29010.4067/S0717-95022004000400008Crossref Google Scholar, 17Naidu R.A. Ingle C.J. Deom C.M. Sherwood J.L. The two envelope membrane glycoproteins of tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases.Virology. 2004; 319 (14967492): 107-11710.1016/j.virol.2003.10.012Crossref PubMed Scopus (17) Google Scholar18Tateno H. Uchiyama N. Kuno A. Togayachi A. Sato T. Narimatsu H. Hirabayashi J. A novel strategy for mammalian cell surface glycome profiling using lectin microarray.Glycobiology. 2007; 17 (17693441): 1138-114610.1093/glycob/cwm084Crossref PubMed Scopus (138) Google Scholar), by lectin microarray analysis using 96 lectins (19Tateno H. Toyota M. Saito S. Onuma Y. Ito Y. Hiemori K. Fukumura M. Matsushima A. Nakanishi M. Ohnuma K. Akutsu H. Umezawa A. Horimoto K. Hirabayashi J. Asashima M. Glycome diagnosis of human induced pluripotent stem cells using lectin microarray.J. Biol. Chem. 2011; 286 (21471226): 20345-2035310.1074/jbc.M111.231274Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 20Tateno H. Kuno A. Itakura Y. Hirabayashi J. A versatile technology for cellular glycomics using lectin microarray.Methods Enzymol. 2010; 478 (20816480): 181-19510.1016/S0076-6879(10)78008-3Crossref PubMed Scopus (38) Google Scholar). Taken together, RDE (II) was found to have the specific characteristics necessary to modulate the glycan on IgE, when compared with IgG. We previously generated the plasmid vector coding the antibody gene of anti-HA IgG and anti-HA IgE (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). Surprisingly, even the variable regions were conserved, wherein anti-HA IgE was not able to neutralize the influenza virus in vitro (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). For the neutralizing assay, the specimens were treated with RDE (II) (13Tyrrell D.A. Horsfall Jr., F.L. A procedure which eliminates nonspecific inhibitor from human serum but does not affect specific antibodies against influenza viruses.J. Immunol. 1952; 69 (13011314): 563-574PubMed Google Scholar), followed by incubation with influenza virus in the presence of trypsin, which cleaves the HA of the influenza virus (21Klenk H.D. Rott R. Orlich M. Blödorn J. Activation of influenza A viruses by trypsin treatment.Virology. 1975; 68 (173078): 426-43910.1016/0042-6822(75)90284-6Crossref PubMed Scopus (549) Google Scholar). We also reconfirmed that the antigen-binding activity of anti-HA IgE treated with RDE (II) was reduced to the background level, although anti-HA IgG was almost not affected (Fig. 1A) (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). Moreover, we were almost unable to detect RDE (II)–treated IgE by antibodies against ε chain, despite RDE (II)–treated anti-HA IgG being detected at almost the same level as the untreated antibody (Fig. 1B). In our previous report (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar), we could not detect RDE (II)–treated anti-HA IgE using antibodies against ε chain by Western blotting. However, the light chain of anti-HA IgG and anti-HA IgE was expressed from the same plasmid vector. Therefore, we measured the binding activity of light chain-binding protein (BP) (Fig. 1C). We detected bands of untreated and RDE (II)–treated IgE at over 250 kDa, which was much larger than predicted (around 200 kDa) (Fig. 1C) (3Balbino B. Conde E. Marichal T. Starkl P. Reber L.L. Approaches to target IgE antibodies in allergic diseases.Pharmacol. Ther. 2018; 191 (29909239): 50-6410.1016/j.pharmthera.2018.05.015Crossref PubMed Scopus (32) Google Scholar). Taken together, these results suggest that RDE (II) affects not only the antigen-binding region of anti-HA IgE but also the constant region, except for the light chain. Because anti-HA IgG was not affected by RDE (II), it is also suggested that the reactivity of RDE (II) is specific for anti-HA IgE. To confirm whether RDE (II) affected the IgE antibody, purified mouse monoclonal IgE (anti-TNP IgE) and IgG (anti-T-2 mycotoxin IgG) were treated with RDE (II) and detected by Western blotting. Although RDE (II)–treated IgG was detected as several smaller bands, a single band over 150 kDa, which was the intact size of IgG antibodies (Fig. 2A, lanes 5–8), was detected and identified as the untreated IgG (Fig. 2A, lanes 1–4). In contrast, in the absence of RDE (II), the band of the purified IgE was mainly detected as over 250 kDa, as seen in Fig. 1C (Fig. 2A, lanes 9–12). We also detected a much smaller-sized band of RDE (II)–treated IgE, around 150 kDa (Fig. 2A, lanes 13–16). Moreover, we obtained the same results by CBB staining (Fig. 2B) and light chain BP (Fig. 2C). To analyze which heavy chain (ε chain) or light chain RDE (II) has a greater effect, we analyzed RDE (II)–treated IgE under reducing conditions. Using antibodies against the ε chain, we obtained a smaller band (around 50 kDa) than that of the untreated (over 75 kDa), depending on the serial dilution (Fig. 2D). Using light chain BP, the band size obtained was the same even after treating with RDE (II) (Fig. 2E). These results indicate that RDE (II) affects the ε chain and not the light chain, which is similar to that demonstrated in Fig. 1C. To confirm the influence of RDE (II) on the constant region, we quantified the binding activity of the antibodies against the constant region in the presence and absence of RDE (II). In the plate coated with anti-mouse immunoglobulins (Igs), RDE (II)–treated IgE levels decreased depending on the concentration of RDE (II) (Fig. 2F), although RDE (II)–treated IgG was stably detected at almost the same level as that of the untreated (Fig. 2G). Moreover, a 34-fold dilution was the titer of RDE (II) for IgE in the reduction of binding levels to coated anti-mouse Igs (Fig. 2F) and that of the band from over 75 to 50 kDa (Fig. 2, D and F). Taken together, these results suggest that the reduction of the size by RDE (II) correlates with the binding activity to antibodies against ε chain. Next, we confirmed whether RDE (II) could reduce the binding activity of serum IgE to anti-ε chain ex vivo. We previously succeeded in inducing the expression of anti-HA IgE in the mouse serum by hydrodynamic injection with the plasmid vector encoding the antibody gene (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). We obtained serum from mice in which anti-HA IgE and anti-HA IgG antibody coding gene had been transferred, followed by incubation with RDE (II). The expression level of RDE (II)–treated anti-HA IgE was not detected in the serum at all, although 2400 ng/ml (= 103.4) of the untreated specimen was detected (Fig. 3A). On the contrary, RDE (II)–treated anti-HA IgG in serum was detected at the same level as that in the absence of RDE (II) (Fig. 3B). We also confirmed that the entire level of IgE in the mouse serum was reduced by treatment with RDE (II) (Fig. 3C). In contrast, the entire IgG level was the same as the level both of RDE (II)–treated and -untreated specimens (Fig. 3D). These results indicate that RDE (II) specifically reduces the binding of IgE to anti-ε antibodies but does not affect the level of IgG in serum. Therefore, this suggests that RDE (II) influences the construction of the ε chain and not the γ chain. RDE (II) treatment affects the construction of IgE. Therefore, we investigated whether RDE (II) alters IgE-mediated mast cell activation. To evaluate the stimulation and release of cytokines and histamines from BMMCs with RDE (II)–treated IgE, BMMCs were incubated with RDE (II)–treated anti-dinitrophenol (DNP) IgE (22Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. Klinman N.R. Katz D.H. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.J. Immunol. 1980; 124 (7373045): 2728-2737Crossref PubMed Google Scholar). Furthermore, BMMCs were activated by cross-linking IgE with DNP–human serum albumin (HSA). The supernatants were obtained, and the induction levels of TNFα, IL-6, and histamine were measured, which are the classical activation markers of mast cells. Although untreated IgE-sensitized BMMCs released large amounts of TNFα, IL-6, and histamine, all of the levels were significantly reduced in RDE (II)–treated IgE-sensitized BMMCs (Fig. 4, A–C). To confirm the binding of RDE (II)–treated IgE to BMMCs, we sensitized the cells with untreated or RDE (II)–treated anti-DNP IgE. As shown in Fig. 4D, although untreated IgE could bind to BMMCs, RDE (II)–treated IgE could barely bind to BMMCs (Fig. 4D). However, the binding level of control IgG to bone marrow–derived macrophages (BMMs) was barely affected by RDE (II). We also confirmed that RDE (II) was not toxic for BMMCs, because propidium iodide–positive cells were barely detected in BMMCs with RDE (II)–treated IgE (Fig. S1). These data indicate that RDE (II)–treated IgE can no longer bind to BMMCs. Therefore, BMMCs are unable to release cytokines and histamine even in presence of the antigen. To evaluate whether RDE (II)–treated anti-DNP IgE has a similar effect in vivo, we examined IgE-mediated passive cutaneous anaphylaxis (PCA) in mouse models. Increased vascular permeability was observed in mice that were injected with untreated anti-DNP IgE (Fig. 4, E and F). On the contrary, vascular permeability was significantly decreased in mice with RDE (II)–treated anti-DNP IgE, as in the control (Fig. 4, E and F). These results correlated with those observed in the experimental data of BMMC. RDE (II) significantly affects the structure and activity of IgE. Therefore, this suggests that the neutralizing titer of anti-HA IgE against influenza virus was reduced in vitro, compared with that of anti-HA IgG in our previous report (14Yamazaki T. Nagashima M. Ninomiya D. Ainai A. Fujimoto A. Ichimonji I. Takagi H. Morita N. Murotani K. Hasegawa H. Chiba J. Akashi-Takamura S. Neutralizing antibodies induced by gene-based hydrodynamic injection have a therapeutic effect in lethal influenza infection.Front. Immunol. 2018; 9 (29416543): 4710.3389/fimmu.2018.00047Crossref PubMed Scopus (8) Google Scholar). To cleave the HA of influenza virus to enable the virus to infect host cells, we incubated the cells in the presence of trypsin (21Klenk H.D. Rott R. Orlich M. Blödorn J. Activation of influenza A viruses by trypsin treatment.Virology. 1975; 68 (173078): 426-43910.1016/0042-6822(75)90284-6Crossref PubMed Scopus (549) Google Scholar). We also confirmed the binding activity of the anti-ε chain to the trypsin-treated IgE. Although trypsin-treated IgG was detected at almost the same level as intact IgG (Fig. S2B), trypsin-treated IgE was significantly reduced by 2-fold (Fig. S2A). To evaluate whether trypsin affected the structure of IgE, we separated trypsin-treated IgE by Western blotting under nonreducing conditions. Interestingly, the main band of the IgE was reduced to around 150 kDa, as seen previously in RDE (II)–treated IgE (Fig. S2C, lanes 10–12). On the contrary, trypsin-treated IgG was detected at the same size as untreated IgG (Fig. S2C, lanes 4–6). Under reducing conditions, we also obtained a smaller band, around 50 kDa, than that of untreated IgE, depending on the serial dilution (Fig. S2D). These results suggest that trypsin also specifically affects the structure of IgE as RDE (II). Both RDE (II) and trypsin significantly reduced the binding activity of IgE against anti-ε chain antibodies and affected the structure. It was considered that RDE (II) has protease activity like trypsin. To confirm our hypothesis, we used preincubated RDE (II) at 56 or 100 °C (56 °C-RDE (II) or 100 °C-RDE (II)), followed by treatment of IgE and IgG. 56 °C-RDE (II)–treated IgE indicated the same binding activity to anti-ε chain as intact RDE (II), which was reduced by 2-fold compared with the control (Fig. 5A). However, 100 °C-RDE (II)–treated IgE was not detected in the reduction of the binding activity to the anti-ε chain at the same level as the control (Fig. 5A). We also found that a band of ∼150 kDa was not detected in 100 °C-RDE (II)–treated IgE, and only a band over 250 kDa was detected (Fig. 5B). These results suggest that the activity of RDE (II) is caused by a function of an enzyme such as protease or glycosidase. To remove the possibility of low molecular weight compound-induced function, we dialyzed RDE (II) to exclude any molecule below 12–16 kDa. We confirmed that the band of IgE treated with dialyzed RDE (II) was also around 150 kDa, the same as that treated with intact RDE (II) (Fig. 5C). These results suggest that RDE (II) works as an enzyme for the modulation of IgE. Then, we analyzed the time course of the reduction by treatment with RDE (II). After incubation for 10 min, ladder bands between >250 and ∼150 kDa were detected (Fig. 5D, lane 2). The band over 250 kDa reduced and that around 150 kDa increased over time (Fig. 5D, lanes 3 and 4)" @default.
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• W2920252492 date "2019-04-01" @default.
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• W2920252492 title "Receptor-destroying enzyme (RDE) from Vibrio cholerae modulates IgE activity and reduces the initiation of anaphylaxis" @default.
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