The Journal of Immunology Cloning, Expression, and Mapping of Allergenic Determinants of ␣S1-Casein, a Major Cow’s Milk Allergen1 Ulrike Schulmeister,* Heidrun Hochwallner,† Ines Swoboda,†‡ Margarete Focke-Tejkl,†‡ Beate Geller,* Mats Nystrand,§ Annika Ha¨rlin,§ Josef Thalhamer,¶ Sandra Scheiblhofer,¶ Walter Keller,ʈ Bodo Niggemann,# Santiago Quirce,** Christoph Ebner,†† Adriano Mari,‡‡ Gabrielle Pauli,§§ Udo Herz,¶¶ Rudolf Valenta,2†‡ and Susanne Spitzauer* Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow’s milk allergen, ␣S1-casein, from a bovine mammary gland cDNA library with allergic patients’ IgE Abs. Recombinant ␣S1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of ␣S1-casein were determined with recom- binant fragments and synthetic peptides spanning the ␣S1-casein sequence using microarrayed components and sera from 66 cow’s milk-sensitized patients. The allergenic activity of r␣S1-casein and the ␣S1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human Fc␧RI, which had been loaded with the patients’ serum IgE. Our results demonstrate that r␣S1-casein as well as ␣S1-casein-derived peptides exhibit IgE reactivity, but mainly the intact r␣S1-casein induced strong basophil degranulation. These results suggest that primarily intact ␣S1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant ␣S1-casein as well as ␣S1-casein-derived pep- tides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy. The Journal of Immunology, 2009, 182: 7019–7029. mmunoglobulin E-mediated allergies belong to the most diet and therefore represents one of the most important food al- common forms of immunologically mediated forms of hy- lergen sources in terms of frequency and severity of allergic man- I persensitivity reactions to food (1). In sensitized individuals ifestations (6–9). The symptoms of cow’s milk allergy are due to dietary intake of food can cause a variety of clinical manifestations IgE-mediated activation of mast cells and basophils as well as to reaching from oral allergy syndrome and gastrointestinal symp- activation of allergen-specific T cells, and they comprise a toms (e.g., vomiting, diarrhea) to skin, respiratory, and severe sys- plethora of gastrointestinal, skin, respiratory, and severe sys- temic manifestations such as anaphylactic shock (2–4). The de- temic manifestations such as death due to anaphylactic shock. velopment of food allergy shows a typical course (5). It starts early Unlike in respiratory allergy, which proceeds untreated from in childhood mainly against Ags encountered in the initial diet mild (e.g., rhinoconjunctivitis) to severe manifestations (e.g., (e.g., cow’s milk, eggs) and affects between 4% and 6% of chil- asthma), many milk-allergic children grow out their allergy, and dren. Milk is one of the first food components introduced into the the induction of tolerance against cow’s milk allergens has al- ready been described (10, 11). In this context it has been re- ϩ ϩ *Institute of Medical and Chemical Laboratory Diagnostics, †Department of Patho- ported that allergen-specific CD4 CD25 regulatory T cells physiology, Division of Immunopathology, Center for Physiology and Pathophysiol- may be involved in the induction of tolerance against cow’s ogy, and ‡Christian Doppler Laboratory for Allergy Research, Medical University of milk allergens in children who outgrew cow’s milk allergy (12). Vienna, Austria; §Phadia, Uppsala, Sweden; ¶Department of Molecular Biology, Di- vision of Allergy and Immunology, University of Salzburg, Austria; ʈInstitute for Furthermore, it has been suggested that the development of tol- Chemistry, Structural Biology, Karl-Franzens-University, Graz, Austria; #Department erance to cow’s milk allergens is associated with a reduction of of Pediatric Allergology and Pneumology, Hedwig-von-Rittberg-Zentrum, German allergen-specific IgE levels and a reduction of IgE recognition of Red Cross Hospital Westend, Berlin, Germany; **Hospital La Paz, Allergy Depart- ment, Madrid, Spain; ††Allergy Clinic Reumannplatz, Vienna, Austria; ‡‡Division of certain sequential epitopes (13, 14). Reduced sensitivities in the Immunopathology, Department of Pathophysiology, Center for Clinical and Experi- gut and the outcome of oral challenge tests are also reflected by mental Allergy, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; §§Service de Pneumologie, Hoˆpitaux Universita- reduced skin sensitivity to food allergens (15, 16). ires, Strasbourg, France; and ¶¶Mead Johnson Nutritionals, Evansville, IN 47712 Cow’s milk contains Ͼ25 different proteins, but only the whey Received for publication August 13, 2007. Accepted for publication March 23, 2009. proteins ␣-lactalbumin, ␤-lactoglobulin, BSA, and lactoferrin, as The costs of publication of this article were defrayed in part by the payment of page well as the four caseins, have been identified as allergens (17). The charges. This article must therefore be hereby marked advertisement in accordance casein fraction is composed of ␣S1-, ␣S2-, ␤-, and ␬-casein, of with 18 U.S.C. Section 1734 solely to indicate this fact. which ␣S1-casein seems to be a major allergen according to IgE 1 This work was supported by Grants F01804, F0105, and F01815 from the Austrian and T cell recognition data (18–21). Science Foundation and by a research grant from Biomay, Vienna, Austria. Here we constructed an expression cDNA library from bo- 2 Address correspondence and reprint requests to Dr. Rudolf Valenta, Christian Dopp- ler Laboratory for Allergy Research, Division of Immunopathology, Department of vine mammary glands and used IgE Abs from cow’s milk-al- Pathophysiology, Center of Physiology, Pathophysiology and Immunology, Medical lergic patients to isolate a cDNA coding for ␣S1-casein and University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria. E-mail IgE-reactive ␣S1-casein fragments. The recombinant ␣S1-ca- address: [email protected] sein allergen was obtained by expression in Escherichia coli, Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 purified, and characterized regarding its physicochemical and www.jimmunol.org/cgi/doi/10.4049/jimmunol.0712366 7020 CLONING AND MAPPING OF ALLERGENIC DETERMINANTS OF ␣S1-CASEIN Table I. Characterization of milk-allergic patientsa Age Allergy Milk-Related CMA Total r␣S1-Casein Patient Age Started Sex Country Symptoms SPT CM Challenge Outgrown Other Allergies IgE (kU/L) CM sIgE (kUA/L) (FI) 1* 61 yr NK F A NK Pos ND NK Cat, WF, T, K, P 355 26 21.0 2* 64 yr 38 yr F F U, Sys, GI Pos Pos No No 1,302 209.6 1,288.0 3* 42 yr NK F A OAS Neg ND No Mite, cat 148 15.3 17.5 4* 26 yr NK M G NK ND Pos NK NK 3,736 5.34 82.5 5 17 yr NK F G NR ND Negd NA NK 926 4.12 29.0 6* 16 yr NK F G NR ND Negd NA HE 433 12.4 4513.0 7* 14 yr NK F G NK ND NK NK NK 14,525 40.2 12,058.5 8* 13 yr NK M G RC, V ND Pos NK HE 866 7.8 210.0 9* 13 yr 11 mo M I U, E, V, AS Pos NPc No PO, HE 909 147.4 12,444.5 10 11 yr NK M G NK ND NK NK NK 763 4.99 195.0 11* 11 yr 9 mo F I U, AE, AS Pos NPc No Candida ND 22.5 8,717.0 12* 10 yr NK F G Ap Pos Pos NK ND 1,432 44.8 3,785.5 13 10 yr NK F G U, V, D Pos Pos NK ND 653 10.4 893.0 14* 8 yr NK M G U,I Pos Posd NK HE 455 3.77 973.0 15* 8 yr NK M G E, U, AS Pos Posd NK HE 2,200 49.7 6,165.0 16 8 yr NK M G U, R, RC Pos Pos NK HE 1271 84.1 22,097.5 17* 7 yr NK M G U, R, AS Pos Posd NK HE 399 17.8 836.5 18* 7 yr NK M G NK ND NK NK NK 2,275 20 4,931.5 19 7 yr NK M G Sys Pos Posd NK NK 7,480 101 2,435.5 20* 6 yr NK NK G NK ND NK NK NK 246 6.09 356.0 21 6 yr NK M G NK ND NK NK NK 297 5.95 108.0 22* 5 yr NK M G NR ND Negd NA HE 866 4.35 517.5 23 5 yr NK F A U, AS Pos ND NK GP, mite, cat, HE, PN 386 16.5 319.0 24 5 yr NK M G NK ND NK NK NK 252 27.9 1,595.0 25* 5 yr NK M G NK ND NK NK NK 2,760 6.44 1,000.5 26 5 yr NK M G NK ND NK NK NK 848 45.2 17.5 27* 4 yr NK M G U, I ND Posd NK HE 56.6 6.19 3,658.5 28* 4 yr NK F G Sys Pos Posd NK NK 974 98 19,178.0 29* 4 y NK M G NK ND NK NK NK 246 6.4 117.5 30* 4 y NK M G NK ND NK NK NK 1,894 3.81 87.5 31* 4 y NK F G Sys Pos Pos NK HE 489 62.5 8,847.5 32* 4 yr NK F G NK ND NK NK NK 116 6.94 943.0 33 3 yr NK M G U Pos Pos NK ND 137 24.6 3,986.5 34* 3 yr NK M G U, AE, R ND Posd NK HE 59.1 4.13 27.5 35* 3 yr NK M G U, R, W Pos Posd NK HE 59.1 4.13 123.0 36 3 yr NK M G NR ND Neg NA NK 10.9 5.81 1,025.5 37* 3 yr NK F G Sys Pos Posd NK NK 125 11 1,474.0 38* 3 yr NK M G NK ND NK NK NK 26 5.19 61.5 39* 3 yr NK F G NK ND NK NK NK 1,718 4.15 2,480.0 40* 3 yr NK M G Sys ND ND NK NK 325 44.4 5,523.0 41* 3 yr NK F G NK ND NK NK NK 125 11 591.5 42* 3 yr NK F G NK ND NK NK NK 201 9.66 766.5 43* 3 yr NK M G NK ND NK NK NK 67.5 10.7 142.5 44* 2 yr NK M G RC, U, OAS Pos Posd NK ND 1,466 82.7 36.5 45* 2 yr 9 mo F S U (face) Pos Posb Yes HE ND 3.82 28.0 46* 2 yr 3 mo F S U, AD, V Pos Posb No HE, fish ND 68.4 21,518.0 47* 2 yr NK M A AD, OB Neg ND NK Mite 134 9.3 620.0 48 2 yr NK F G NK ND NK NK NK 53.9 6.71 67.0