BACKGROUND: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate. METHODS: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles. CONCLUSIONS: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate.

Biochemistry Section Faculty of Sciences and Chemical Technologies Universidad de Castilla La Mancha Ave Camilo José Cela 10 13071 Ciudad Real Spain

Centre for Kode Technology Innovation School of Engineering Computer and Mathematical Sciences Faculty of Design and Creative Technologies Auckland University of Technology Auckland New Zealand

Departamento de Bioquímica Microbiología Biología Celular y Genética Área de Microbiología Universidad de La Laguna Entrada Campus Anchieta 4 38200 La Laguna Tenerife Canary Islands Spain

Department of Medical Biology Faculty of Science University of South Bohemia in České Budějovice Branišovská 31 37005 České Budějovice Czechia

Department of Veterinary Pathobiology Center for Veterinary Health Sciences Oklahoma State University Stillwater OK 74078 USA

Institute of ParasitologyBiology Centre Czech Academy of Sciences Branišovská 31 37005 České Budějovice Czechia

National Medical Research Center for Obstetrics Gynecology and Perinatology Named After Academician 5 1 Kulakov Oparina str 4 117198 Moscow Russian Federation

SaBio Instituto de Investigación en Recursos Cinegéticos IREC CSIC UCLM JCCM Ronda de Toledo s n 13005 Ciudad Real Spain

Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences Miklukho Maklaya str 16 10 117997 Moscow Russian Federation

UMR BIPAR INRAE ANSES Ecole Nationale Vétérinaire d'Alfort Université Paris Est 94700 Maisons Alfort France

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Van Nunen SA, O’Connor KS, Clarke LR, Boyle RX, Fernando SL. An association between tick bite reactions and red meat allergy in humans. Med J Aust. 2009;190:510–511. doi: 10.5694/j.1326-5377.2009.tb02533.x. PubMed DOI

Mullins RJ, James H, Platts-Mills TAE, Commins S. Relationship between red meat allergy and sensitization to gelatin and galactose-α-1,3-galactose. J Allergy Clin Immunol. 2012;129:1334–42.e1. doi: 10.1016/j.jaci.2012.02.038. PubMed DOI PMC

Çelebioğlu E, Akarsu A, Şahiner ÜM. IgE-mediated food allergy throughout life. Turk J Med Sci. 2021;51:49–60. doi: 10.3906/sag-2006-95. PubMed DOI PMC

Ramasamy R. Mosquito vector proteins homologous to α1-3 galactosyl transferases of tick vectors in the context of protective immunity against malaria and hypersensitivity to vector bites. Parasit Vectors. 2021;14:303. doi: 10.1186/s13071-021-04801-7. PubMed DOI PMC

Galili U. Evolution in primates by “Catastrophic-selection” interplay between enveloped virus epidemics, mutated genes of enzymes synthesizing carbohydrate antigens, and natural anti-carbohydrate antibodies. Am J Phys Anthropol. 2019;168:352–363. doi: 10.1002/ajpa.23745. PubMed DOI

Kersh GJ, Salzer J, Jones ES, et al. Tick bite as a risk factor for alpha-gal-specific immunoglobulin E antibodies and development of alpha-gal syndrome. Ann Allergy Asthma Immunol. 2023;130:472–478. doi: 10.1016/j.anai.2022.11.021. PubMed DOI PMC

Sharma SR, Karim S. Tick saliva and the alpha-gal syndrome: finding a needle in a haystack. Front Cell Infect Microbiol. 2021;11:680264. doi: 10.3389/fcimb.2021.680264. PubMed DOI PMC

Carson AS, Gardner A, Iweala OI. Where’s the beef? Understanding allergic responses to red meat in Alpha-Gal Syndrome. J Immunol. 2022;208:267–277. doi: 10.4049/jimmunol.2100712. PubMed DOI PMC

Cabezas-Cruz A, Hodžić A, Román-Carrasco P, Mateos-Hernández L, Duscher GG, Sinha DK, et al. Environmental and molecular drivers of the α-Gal syndrome. Front Immunol. 2019;10:1210. doi: 10.3389/fimmu.2019.01210. PubMed DOI PMC

Saretta F, Giovannini M, Mori F, Arasi S, Liotti L, Pecoraro L, et al. Alpha-Gal Syndrome in children: peculiarities of a “tick-borne” allergic disease. Front Pediatr. 2021;9:801753. doi: 10.3389/fped.2021.801753. PubMed DOI PMC

Chandrasekhar JL, Cox KM, Erickson LD. B Cell responses in the development of mammalian meat allergy. Front Immunol. 2020;11:1532. doi: 10.3389/fimmu.2020.01532. PubMed DOI PMC

Apostolovic D, Rodrigues R, Thomas P, Starkhammar M, Hamsten C, van Hage M. Immunoprofile of α-Gal- and B-antigen-specific responses differentiates red meat-allergic patients from healthy individuals. Allergy. 2018;73:1525–1531. doi: 10.1111/all.13400. PubMed DOI

Apostolovic D, Mihailovic J, Commins SP, Wijnveld M, Kazimirova M, Starkhammar M, et al. Allergenomics of the tick Ixodes ricinus reveals important α-Gal-carrying IgE-binding proteins in red meat allergy. Allergy. 2020;75:217–220. doi: 10.1111/all.13978. PubMed DOI PMC

Chung CH, Chan E, Morse M, Hosen J, Zhou Q, Hicklin DJ. Cetuximab-induced anaphylaxis and IgE specific for galactose-α-1,3-galactose. New England J Med. 2008;358:1109–1117. doi: 10.1056/NEJMoa074943. PubMed DOI PMC

Cabezas-Cruz A, Espinosa PJ, Alberdi P, Šimo L, Valdés JJ, Mateos-Hernánde L, et al. Tick galactosyltransferases are involved in α-Gal synthesis and play a role during Anaplasma phagocytophilum infection and Ixodes scapularis tick vector development. Sci Rep. 2018;8:14224. doi: 10.1038/s41598-018-32664-z. PubMed DOI PMC

Sharma SR, Crispell G, Mohamed A, Cox C, Lange J, Choudhary S, et al. Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-Galactosidase and β-1,4 Galactosyltransferase enzymes in α-Gal metabolism. Front Cell Infect Microbiol. 2021;11:775371. doi: 10.3389/fcimb.2021.775371. PubMed DOI PMC

Vora A, Taank V, Dutta SM, Anderson JF, Fish D, Sonenshine DE, et al. Ticks elicit variable fibrinogenolytic activities upon feeding on hosts with different immune backgrounds. Sci Rep. 2017;7:44593. doi: 10.1038/srep44593. PubMed DOI PMC

Nuttall PA. Wonders of tick saliva. Ticks Tick-borne Dis. 2019;10:470–481. doi: 10.1016/j.ttbdis.2018.11.005. PubMed DOI

Villar M, Pacheco I, Merino O, Contreras M, Mateos-Hernández L, Padro E, et al. Tick and host derived compounds detected in the cement complex substance. Biomolecules. 2020;10:E555. doi: 10.3390/biom10040555. PubMed DOI PMC

Neelakanta G, Sultana H. Tick saliva and salivary glands: what do we know so far on their role in arthropod blood feeding and pathogen transmission. Front Cell Infect Microbiol. 2022;11:816547. doi: 10.3389/fcimb.2021.816547. PubMed DOI PMC

Zhou J, Gong H, Zhou Y, Xuan X, Fujisaki K. Identification of a glycine-rich protein from the tick Rhipicephalus haemaphysaloides and evaluation of its vaccine potential against tick feeding. Parasitol Res. 2006;100:77–84. doi: 10.1007/s00436-006-0243-7. PubMed DOI

Vaz-Rodrigues R, Mazuecos L, de la Fuente J. current and future strategies for the diagnosis and treatment of the Alpha-Gal Syndrome (AGS) J Asthma Allergy. 2022;15:957–970. doi: 10.2147/JAA.S265660. PubMed DOI PMC

Villar M, Pacheco I, Mateos-Hernández L, et al. Characterization of tick salivary gland and saliva alphagalactome reveals candidate alpha-gal syndrome disease biomarkers. Expert Rev Proteomics. 2021;18:1099–1116. doi: 10.1080/14789450.2021.2018305. PubMed DOI

Contreras M, Pacheco I, Alberdi P, Díaz-Sánchez S, Artigas-Jerónimo S, Mateos-Hernández L, et al. Allergic reactions and immunity in response to tick salivary biogenic substances and red meat consumption in the zebrafish model. Front Cell Infect Microbiol. 2020;10:78. doi: 10.3389/fcimb.2020.00078. PubMed DOI PMC

Kageyama R, Fujiyama T, Satoh T, Keneko Y, Kitano S, Tokura Y, et al. The contribution made by skin-infiltrating basophils to the development of alpha gal syndrome. Allergy. 2019;74:1805–1807. doi: 10.1111/all.13794. PubMed DOI

Balla KM, Lugo-Villarino G, Spitsbergen JM, et al. Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants. Blood. 2010;116:3944–3954. doi: 10.1182/blood-2010-03-267419. PubMed DOI PMC

Li CL, Liu N. Expression of interleukin-1β, interleukin-4, interferon-γ and tumour necrosis factor α in different tissue in a dinitrochlorobenzene-induced ear swelling test in mice. Skin Res Technol. 2023;29:e13255. doi: 10.1111/srt.13255. PubMed DOI PMC

KrstićRistivojević M, Apostolović D, Smiljanić K. Enterocytes in food hypersensitivity reactions. Animals. 2021;11:2713. doi: 10.3390/ani11092713. PubMed DOI PMC

Bailone RL, de Aguiar LK, de Oliveira Roca R, Borra RC, Corrêa T, Janke H, Fukushima HCS. Zebrafish as an animal model for food safety research: Trends in the animal research. Food Biotechnol. 2019;33:283–302. doi: 10.1080/08905436.2019.1673173. DOI

Fuentes-Appelgren P, Opazo R, Barros L, Feijoó CG, Urzúa V, Romero J. Effect of the dietary inclusion of soybean components on the innate immune system in zebrafish. Zebrafish. 2014;11:41–49. doi: 10.1089/zeb.2013.0934. PubMed DOI

Macdougall JD, Thomas KO, Iweala OI. The meat of the matter: understanding and managing alpha-gal syndrome. Immunotargets Ther. 2022;11:37–54. doi: 10.2147/ITT.S276872. PubMed DOI PMC

Mashoof S, Criscitiello M. Fish immunoglobulins. Biology. 2016;5:45. doi: 10.3390/biology5040045. PubMed DOI PMC

Kýčková K, Kopecký J. Effect of tick saliva on mechanisms of innate immune response against Borrelia afzelii. J Med Entomol. 2006;43:1208–1214. doi: 10.1093/jmedent/43.6.1208. PubMed DOI

Narasimhan S, Kurokawa C, Diktas H, Strank NO, Černý J, Murfin K, et al. Ixodes scapularis saliva components that elicit responses associated with acquired tick-resistance. Ticks Tick Borne Dis. 2020;11:101369. doi: 10.1016/j.ttbdis.2019.101369. PubMed DOI PMC

Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1:2856–2860. doi: 10.1038/nprot.2006.468. PubMed DOI

Matthews M, Varga ZM. Anesthesia and euthanasia in zebrafish. ILAR J. 2012;53:192–204. doi: 10.1093/ilar.53.2.192. PubMed DOI

Lu Y, Shao A, Shan Y, Zhao H, Leiguo M, Zhang Y, Tang Y, Zhang W, Jin Y, Xu L. A standardized quantitative method for detecting remnant alpha-Gal antigen in animal tissues or animal tissue-derived biomaterials and its application. Sci Rep. 2018;8:15424. doi: 10.1038/s41598-018-32959-1. PubMed DOI PMC

Obukhova P, Tsygankova S, Chinarev A, et al. Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals? [published correction appears in Glycobiology. 2020 May 19;30(6):415]. Glycobiology. 2020;30:395-406. 10.1093/glycob/cwz107 PubMed

Pacheco I, Díaz-Sánchez S, Contreras M, et al. Probiotic bacteria with high Alpha-Gal content protect zebrafish against mycobacteriosis. Pharmaceuticals. 2021;14:635. doi: 10.3390/ph14070635. PubMed DOI PMC

Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human IgG antibody with anti-alpha-galactosyl specificity. J Exp Med. 1984;160:1519–1531. doi: 10.1084/jem.160.5.1519. PubMed DOI PMC

Mateos-Hernández L, Villar M, Moral A, Rodríguez CG, Arias TA, de la Osa V, et al. Tick-host conflict: immunoglobulin E antibodies to tick proteins in patients with anaphylaxis to tick bite. Oncotarget. 2017;8:20630–20644. doi: 10.18632/oncotarget.15243. PubMed DOI PMC

de la Fuente J, Urra JM, Contreras M, Pacheco I, Ferreras-Colina E, Doncel-Pérez E, et al. A dataset for the analysis of antibody response to glycan alpha-Gal in individuals with immune-mediated disorders. F1000Res. 2021;9:1366. 10.12688/f1000research.27495.2 PubMed PMC

Pacheco I, Fernández de Mera IG, Feo Brito F, Gómez Torrijos E, Villar M, Contreras M, et al. Characterization of the anti-α-Gal antibody profile in association with Guillain-Barré syndrome, implications for tick-related allergic reactions. Ticks Tick Borne Dis. 2021;12:101651. 10.1016/j.ttbdis.2021.101651 PubMed

Young I, Prematunge C, Pussegoda K, Corrin T, Waddell L. Tick exposures and alpha-gal syndrome: a systematic review of the evidence. Ticks Tick Borne Dis. 2021;12:101674. doi: 10.1016/j.ttbdis.2021.101674. PubMed DOI

CDC. Alpha-gal syndrome | CDC. Centers for Disease Control and Prevention. Published March 28, 2022. https://www.cdc.gov/ticks/alpha-gal/index.html. Accessed 12 Sep 2022.

Paudel YN, Kumari Y, Abidin SAZ, Othman I, Shaikh MF. Pilocarpine induced behavioral and biochemical alterations in chronic seizure-like condition in adult zebrafish. Int J Mol Sci. 2020;21:2492. doi: 10.3390/ijms21072492. PubMed DOI PMC

Ribeiro JM, Zeidner NS, Ledin K, Dolan MC, Mather TN. How much pilocarpine contaminates pilocarpine-induced tick saliva? Med Vet Entomol. 2004;18:20–24. doi: 10.1111/j.0269-283x.2003.0469.x. PubMed DOI

Park Y, Kim D, Boorgula GD, De Schutter K, Smagghe G, Šimo L, Archer-Hartmann SA, Azadi P. Alpha-Gal and cross-reactive carbohydrate determinants in the N-glycans of salivary glands in the lone star tick. Amblyomma americanum Vaccines. 2020;8:18. doi: 10.3390/vaccines8010018. PubMed DOI PMC

Shilova NV, Navakouski MJ, Huflejt M, et al. Changes in the repertoire of natural antibodies caused by immunization with bacterial antigens. Biochemistry (Mosc) 2011;76:862–866. doi: 10.1134/S0006297911070170. PubMed DOI

Ricci Hagman J, Westman JS, Hellberg Å, Olsson ML. An update on the GLOB blood group system (and former GLOB collection) Immunohematology. 2018;34:161–163. doi: 10.21307/immunohematology-2018-026. PubMed DOI

Okajima T, Nakamura Y, Uchikawa M, et al. Expression cloning of human globoside synthase cDNAs. Identification of beta 3Gal-T3 as UDP-N-acetylgalactosamine:globotriaosylceramide beta 1,3-N-acetylgalactosaminyltransferase. J Biol Chem. 2000;275:40498–40503. doi: 10.1074/jbc.M006902200. PubMed DOI

Thuresson B, Westman JS, Olsson ML. Identification of a novel A4GALT exon reveals the genetic basis of the P1/P2 histo-blood groups. Blood. 2011;117:678–687. doi: 10.1182/blood-2010-08-301333. PubMed DOI

Spitalnik PF, Spitalnik SL. The P blood group system: biochemical, serological, and clinical aspects. Transfus Med Rev. 1995;9:110–122. doi: 10.1016/s0887-7963(05)80050-1. PubMed DOI

Černý J, Lynn G, DePonte K, Ledizet M, Narasimhan S, Fikrig E. Fractionation of tick saliva reveals proteins associated with the development of acquired resistance to Ixodes scapularis. Vaccine. 2020;38:8121–8129. doi: 10.1016/j.vaccine.2020.10.087. PubMed DOI

Villar M, Urra JM, Rodríguez-Del-Río FJ, Artigas-Jerónimo S, Jiménez-Collados N, Ferreras-Colino E, et al. Characterization by quantitative serum proteomics of immune-related prognostic biomarkers for COVID-19 symptomatology. Front Immunol. 2021;12:730710. doi: 10.3389/fimmu.2021.730710. PubMed DOI PMC

Tan KW, Jobichen C, Ong TC, Gao YF, Tiong YS, Wong KN, et al. Crystal structure of Der f 7, a dust mite allergen from Dermatophagoides farinae. PLoS ONE. 2012;7:e44850. doi: 10.1371/journal.pone.0044850. PubMed DOI PMC