Bovine γ‐globulin, lactoferrin, and lactoperoxidase are relevant bovine milk allergens in patients with α‐Gal syndrome

Abbreviations AGS α-Gal syndrome AP Alkaline phosphatase BGG Bovine γ-globulin BSA Bovine serum albumin ELISA Enzyme-linked immunosorbent assay HSA Human serum albumin LF Lactoferrin LPO Lactoperoxidase ROC Receiver operating characteristic RT Room temperature SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis α-Gal Galactose-α-1,3-galactose 1 INTRODUCTION

More than a decade ago, the carbohydrate galactose-α-1,3-galactose (α-Gal) was identified as an IgE epitope that mediates allergic reactions in a novel form of food allergy known as the α-Gal syndrome (AGS).1, 2 The α-Gal epitope is expressed on glycoproteins and glycolipids of non-primate mammals but is also present in some pharmaceuticals. AGS is characterized by late-onset symptoms, usually two to six hours after mammalian meat consumption,3 which is in contrast to common food allergies where the reactions are immediate. The symptoms range from abdominal pain, urticaria to anaphylaxis, and are often severe. Notably, nearly 50% of the patients experience anaphylaxis.4 The syndrome typically develops in middle-aged patients who have previously tolerated mammalian meat.5, 6 The induction of the disease has been shown to be associated with tick bites.7 The first AGS cases were reported in the United States in 2009, shortly followed by reports from Sweden and Australia, and today the syndrome is diagnosed worldwide and its prevalence continues to rise.1, 8, 9

Mammalian meat (beef, pork, and lamb) and especially innards, which are abundant in α-Gal content, are the most common food sources that induce allergic reactions in AGS patients. Other foods that contain α-Gal in lesser amounts are bovine milk and mammalian gelatin.10-12 Many AGS patients experience allergic reactions upon milk and dairy consumption.13 IgE reactivity to bovine milk has been observed in more than 90% in our cohort of 128 AGS patients and in nearly 70% in a US cohort of 261 patients.4, 6 Furthermore, a study on 100 randomly selected AGS patients from a US cohort showed that 33% of the allergic manifestations were triggered by dairy.14 In addition, analysis of a large US cohort of 2500 AGS patients revealed that 10–20% of patients reacted to milk.15 The most commonly reported symptoms to bovine milk were abdominal pain and urticaria with delayed onset.16, 17

To date, no milk glycoproteins or glycolipids that carry α-Gal have been identified. This is in contrast to meat, where proteins that carry α-Gal have been thoroughly investigated.18-20 Kollmann et al have shown that bovine γ-globulin (BGG) is the most prominent carrier of α-Gal and IgE reactive glycoprotein in beef.19 Interestingly, BGG is present in milk as well.

The aim of this study was to elucidate the importance of bovine milk as an α-Gal-containing food source. The focus was to characterize AGS patients’ IgE responses to milk proteins, to identify α-Gal carrying milk glycoproteins, and assess their allergenicity among AGS patients.

2 MATERIALS AND METHODS 2.1 Patient cohort and ethics statement

Thirty-eight patients with IgE to milk were selected from our cohort of patients with diagnosed AGS (Table 1). The selection was randomized among almost all available patients (91.4% have IgE to milk4). A total of 34/38 answered a questionnaire and were interviewed by the same allergologist with many years of experience in food allergy, regarding milk and dairy consumption and tolerance. Patients’ sera were collected and either used individually or as a pool. In addition, three healthy non-atopic and two atopic donors participated in the study as controls. Allergen-specific IgE levels against α-Gal (bovine thyroglobulin), milk, α-lactalbumin, β-lactoglobulin, and caseins were determined by ImmunoCAP (Phadia AB/Thermo Fisher Scientific). IgE antibodies against BGG, LF, LPO, and whey were measured by coupling biotinylated BGG, LF, LPO, and whey proteins to Streptavidin ImmunoCAP (Phadia AB/Thermo Fisher Scientific) according to the manufacturer's instructions. The cutoff for allergen-specific IgE was ≥0.1 kUA/L. The study was approved by the Swedish ethical review authority (Ethical permit No 2011/1604-31/2, 2018/2483-32) and performed in accordance with the declaration of Helsinki. All AGS patients and controls gave their written informed consent.

TABLE 1. Data on AGS patients and their specific IgE levels to α-Gal, beef, milk, and milk proteins determined by ImmunoCAP Patient no. Sex/Age Reactions to mammalian meat Reaction to milk/dairy Specific IgE ImmunoCAP [kUA/L] α-Gal Beef Milk α-LA β-LG Caseins BGG LF LPO Whey 1 M/56 GI, U n.d. 16 9.2 2.4 <0.1 <0.1 <0.1 0.49 1.39 0.18 0.38 2 F/50 AE, U Tolerant 4.9 0.46 0.11 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 3 F/59 AE, U, GI, ANA GI 27.3 15.8 5.57 <0.1 <0.1 0.17 2.9 4.48 0.9 1.7 4 M/34 GI GI 31 1.8 0.31 <0.1 <0.1 <0.1 0.14 0.49 <0.1 <0.1 5 M/53 U Tolerant 26 24 12 <0.1 <0.1 0.68 9 12 3.0 6.7 6 F/42 AE, U, ANA GI, U 23 6.8 1.4 <0.1 <0.1 <0.1 0.74 1.4 0.12 0.31 7 F/65 U, GI, ANA Tolerant 4.9 1.3 0.22 <0.1 <0.1 <0.1 0.16 0.23 <0.1 0.14 8 F/70 U, ANA n.d. 13 3.8 0.55 <0.1 <0.1 <0.1 0.23 0.35 <0.1 0.29 9 F/42 AE, U, GI, ANA GI 44 15 1.7 <0.1 <0.1 <0.1 1 2.8 0.18 0.73 10 M/64 AE, U GI 93 13 2.3 <0.1 <0.1 0.11 1.6 7.1 0.31 1.4 11 M/34 AE, U, GI, ANA GI 35 13 4.2 <0.1 <0.1 0.19 3.4 4.6 0.7 2.5 12 M/63 AE Tolerant 40 1.4 0.31 <0.1 <0.1 <0.1 <0.1 0.12 <0.1 <0.1 13 F/29 AE, U, GI Tolerant 14 4.3 0.46 <0.1 <0.1 <0.1 0.25 1.1 <0.1 0.11 14 F/42 AE, U, ANA Tolerant 95 25 5.6 <0.1 <0.1 0.35 5.2 n.d. n.d. 3.1 15 F/34 U, GI GI 95.2 32.5 5.07 0.1 0.17 0.26 3.1 4.2 0.82 1.4 16 M/52 AE, U, GI, ANA Tolerant 94 9.7 1.5 <0.1 <0.1 <0.1 0.57 n.d. n.d. 0.3 17 F/35 U, GI Tolerant 14 7.6 2.4 <0.1 <0.1 0.14 1.5 1.6 0.43 0.89 18 M/44 AE, U, GI, ANA Tolerant 34 14 3.8 <0.1 <0.1 0.11 2.2 n.d. n.d. 1.3 19 M/57 AE, U, GI, ANA GI, Ua 100 47 9.2 <0.1 <0.1 0.25 5.1 6.9 1.8 3.4 20 F/69 AE, U U 31 8.4 1.1 <0.1 <0.1 n.d. 0.45 0.82 0.19 0.28 21 F/37 AE, U, GI, ANA Tolerant 12 2.8 0.76 <0.1 <0.1 <0.1 0.78 1.1 0.13 0.69 22 F/43 AE, U, GI, ANA Tolerant 22 6 1.2 <0.1 <0.1 <0.1 0.55 0.98 <0.1 0.57 23 M/37 GI GIa 13 1.2 0.26 <0.1 <0.1 <0.1 0.21 0.36 <0.1 0.12 24 F/60 U, GI Tolerant 1.2 0.69 0.11 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 25 F/64 AE, U, GI GI 50 31 10 <0.1 <0.1 0.41 6.9 6.5 2.4 5.7 26 M/62 AE, U, GI, ANA GI, U 28 12 1.1 <0.1 <0.1 <0.1 0.68 0.79 <0.1 0.46 27 F/45 AE, U, ANA GI, U 100 26 2.2 <0.1 <0.1 0.43 5.3 8 2.2 4.7 28 M/60 AE, U, GI, ANA Tolerant 88 34 10 0.21 0.16 0.29 0.68 2.2 0.42 0.61 29 M/67 U, ANA Tolerant 10 3.9 0.88 <0.1 <0.1 <0.1 0.34 0.72 <0.1 0.25 30 F/29 AE, U, GI Tolerant 3.2 0.62 0.16 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 31 M/21 U, GI GI 11 2.6 0.36 <0.1 <0.1 <0.1 0.14 0.37 <0.1 <0.1 32 F/47 U, GI n.d. 43 4.1 0.37 <0.1 <0.1 <0.1 <0.1 0.44 <0.1 <0.1 33 M/54 U, AE n.d. 100 25 1.4 <0.1 <0.1 <0.1 0.63 3.3 0.15 0.46 34 F/66 U, AE, GI GI 21 5.3 0.42 <0.1 <0.1 <0.1 0.13 1.4 <0.1 0.13 35 M/46 GI GI 14 6.2 0.51 <0.1 <0.1 <0.1 0.3 0.52 0.12 0.26 36 F/56 U, AE, ANA U 57 23 2.7 <0.1 <0.1 <0.1 1.1 1.9 0.25 0.95 37 M/49 U, GI, ANA GI 43 13 1.8 <0.1 <0.1 <0.1 0.72 2.2 0.27 0.73 38 M30 GI Tolerant 17 6.2 0.88 <0.1 <0.1 <0.1 0.37 1.2 0.15 0.35 Abbreviations: α-LA, α-lactalbumin; β-LG, β-lactoglobulin; BGG, bovine γ-globulin; LF, lactoferrin; LPO, lactoperoxidase; ANA, anaphylaxis; AE, angioedema; GI, gastrointestinal symptoms; U, urticarial; n.d., not determined. a Experience symptoms at each exposure to dairy products. 2.2 Materials and reagents

Unprocessed milk was obtained from a local farm in Belgrade. Commercially available pasteurized 3% and 1.5% fat milk were purchased from a Swedish dairy company (Arla Foods). Commercial pasteurized milk was homogenized and pasteurized at low temperature (72–75°C for less than 15 s). Whey was prepared from unprocessed milk as follows. Unprocessed milk was centrifuged at 4000 rpm for 20 min at +4°C, and the upper lipid layer was removed. Caseins were precipitated by lowering the pH to 4.6 by adding 0.1 M HCl and removed by centrifugation at 4000 rpm for 10 min. The resulting whey was further defatted by tetrachlorethylene extraction. Defatted whey was dialyzed against ammonium bicarbonate buffer, lyophilized, and subsequently dissolved in PBS. Protein concentration was determined by the BCA method. The protein concentration of pasteurized milks was 34 mg/ml as per manufacturer's information. Throughout the study, ultra-pure water (18 mΩ) prepared with a Smart2Pure 3 Barnstead aqua purification system (Thermo Fisher Scientific Inc.) was used. Unless otherwise stated, all the other chemicals were purchased from Sigma-Aldrich.

2.3 SDS-PAGE and immunoblot

Purified milk proteins, α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, BGG, lactoferrin (LF), lactoperoxidase (LPO), bovine serum albumin (BSA) (1.5 µg each, Sigma-Aldrich), and whey (15 µg) were separated under reducing conditions on SDS-PAGE electrophoresis at TGX gradient precast gels (any kDa, Bio-Rad Laboratories) using a Mini Protean Cell II system (Bio-Rad Laboratories). Proteins were transferred to PVDF membranes (0.2 µm pore size) using a Bio-Rad Turbo sys

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