ORIGINAL ARTICLE ANAPHYLAXIS Two galactose-a-1,3-galactose carrying peptidases from pork kidney mediate anaphylactogenic responses in delayed meat allergy C. Hilger 1 , J. Fischer 2 , K. Swiontek 1 , F. Hentges 3 , C. Lehners 3 , B. Eberlein 4 , M. Morisset 3 , T. Biedermann 4 & M. Ollert 1,5 1 Department of Infection and Immunity, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg; 2 Department of Dermatology, Eberhard Karls University, Tuebingen, Germany; 3 Immunology Allergology Unit, Centre Hospitalier, Luxembourg, Luxembourg; 4 Department of Dermatology and Allergology, Technical University Munich, Munich, Germany; 5 Department of Dermatology and Allergy Center, Odense Research Center for Anaphylaxis, University of Southern Denmark, Odense, Denmark To cite this article: Hilger C, Fischer J, Swiontek K, Hentges F, Lehners C, Eberlein B, Morisset M, Biedermann T, Ollert M. Two galactose-a-1,3-galactose carrying peptidases from pork kidney mediate anaphylactogenic responses in delayed meat allergy. Allergy 2016; 71: 711–719. Keywords Allergen; carbohydrate; galactose-a-1,3- galactose; pork kidney; red meat allergy. Correspondence Christiane Hilger, PhD, Department of Infec- tion and Immunity, Luxembourg Institute of Health, 29, rue Henri Koch, L-4354 Esch- sur-Alzette, Luxembourg. Tel.: +352 26970 258 Fax: +352 26970 390 E-mail: christiane.hilger@lih.lu Accepted for publication 2 January 2016 DOI:10.1111/all.12835 Edited by: Werner Aberer Abstract Background: Serum IgE antibodies directed at galactose-a-1,3-galactose (a-Gal) are associated with a novel form of delayed anaphylaxis occurring upon consumption of red meat or innards. Pork kidney is known as the most potent trigger of this syndrome, but the culprit allergens have not yet been identified. The aim of this study was the identification and characterization of pork kidney proteins mediating delayed anaphylactic reactions through specific IgE to a-Gal. Methods: A cohort of 59 patients with specific IgE to a-Gal was screened by immunoblot for IgE-reactive proteins in pork kidney. Proteins were identified by peptide mass fingerprinting. Isolated proteins were assayed in ELISA and ELISA inhibition, basophil activation and skin prick test. Results: Several IgE-binding proteins of high molecular weight (100– >200 kDa) were detected in pork kidney extracts by immunoblot using patient sera and an anti-a-Gal antibody. Two major IgE-binding proteins were identified as porcine angiotensin-I-converting enzyme (ACE I) and aminopeptidase N (AP-N). Reac- tivity of patient sera and anti-a-Gal antibody to both proteins was abolished by carbohydrate oxidation. The a-Gal IgE epitopes were resistant to heat denatura- tion. Pork kidney extract, isolated ACE I, and AP-N were able to activate patient basophils and elicit positive responses in skin prick tests. Conclusion: Two cell-membrane proteins carrying a-Gal epitopes were identified in pork kidney. For the first time, isolated meat proteins were shown to induce basophil activation in patients with delayed anaphylaxis to red meat providing further confirmation for the clinical relevance of these a-Gal-carrying proteins. Delayed anaphylaxis to red meat has been associated with specific IgE antibodies to the carbohydrate epitope galactose- alpha-1,3-galactose (a-Gal) (1). Increasing numbers of cases are reported worldwide and there is strong evidence that tick bites constitute the major sensitization source (2). The pres- ence of specific IgE antibodies to a-Gal constitutes an impor- tant health issue as patients are not only at risk of delayed anaphylaxis to red meat and gelatin-containing products (3–5), but even more importantly to immediate type anaphy- laxis to therapeutic antibodies of rodent origin (6) or to bovine-derived gelatin colloids administered as plasma substi- tutes (5, 7). The use of bioprosthetic material such as heart valves of bovine and porcine origin may put highly sensitized patients at risk of peri- or postoperative hypersensitivity reactions (8). Among other epitopes, a-Gal is known to constitute a major obstacle to xenotransplantation and differ- ent approaches exist to inactivate or mask these epitopes in xenogeneic tissues (9). Abbreviations a-Gal, galactose-a-1,3-galactose; ACE I, angiotensin-I-converting enzyme; AP-N, aminopeptidase N. Allergy 71 (2016) 711–719 © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 711 Allergy