f76 nBos d 4 α-lactalbumin, Milk

Allergen

Cow's milk is popularly consumed throughout the world. Allergy to cow's milk is prevalent among infants and young children during the first year of life, affecting 0.5-3% at the age of 1 year. Bos d 4 (α-lactalbumin) has been reported as a major allergenic protein found in cow's milk whey fraction. The prevalence of sensitization to Bos d 4 among cow’s milk allergy (CMA) patients varies up to 80%. Milk whey proteins like Bos d 4 are heat-labile and therefore are likely to be destroyed after extensive cooking and baking of milk. Thus, specific immunoglobulin E to Bos d 4 might help assess tolerance to cooked or baked milk. Clinical manifestations of Bos d 4-mediated allergy majorly include anaphylaxis and atopic dermatitis. Cross-reactivity has been observed between antibodies against bovine Bos d 5 (β-lactoglobulin) and Bos d 4 in cow's milk due to the shared amino acid sequence. Bos d 4, present in cow and human milk, was also found to be cross-reactive. Moreover, Bos d 4 from cow's milk has been found to exhibit sequence homology with other mammalian milk proteins (like water buffalo, ewe, goat, pig, donkey, horse, and camel).

Epidemiology

Worldwide distribution

Cow’s milk is popularly consumed worldwide by infants, children, and adults (Muthukumar, Selvasekaran et al. 2020). Allergy to cow’s milk is prevalent among infants and young children, with a high prevalence observed during the first year of life (Kaczmarski, Wasilewska et al. 2013, Vandenplas 2017), affecting 0.5-3% at the age of 1 year (Skripak, Matsui et al. 2007, Flom and Sicherer 2019).

Bos d 4 (α-lactalbumin; ALA), Bos d 5 (β-lactoglobulin, BLG) and Bos d 8 (casein) are considered major allergens present in cow’s milk (Villa, Costa et al. 2018). According to World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow’s Milk Allergy (DRACMA) Guidelines, the prevalence of allergic reactions towards Bos d 4 among cow’s milk allergy (CMA) patients ranges between 0 and 80% (Fiocchi, Brozek et al. 2010).

A Taiwan-based study reported sensitization towards Bos d 4 among 60% (114/190) of atopic individuals with specific immunoglobulin E (sIgE) towards whole cow’s milk. Among all three milk allergens (Bos d 4, Bos d 5 and Bos d 8) tested, the sensitization towards Bos d 4 and the mean serum level of Bos d 4 sIgEs were found to be the highest (Chen, Lee et al. 2014). Furthermore, a Poland-based study conducted among 291 children (2-14 years) with a history of CMA also reported sensitization towards Bos d 4 in 18.8% (13/69) of children (Kaczmarski, Wasilewska et al. 2013).

A study in Iran reported allergy against Bos d 4 in 78.3% of patients with CMA (n=86) (Shokouhi Shoormasti, Fazlollahi et al. 2016). Furthermore, a study in Italy conducted on 104 children with suspected CMA (n=58) found the prevalence of IgE reactivity towards Bos d 4 to be 27.6% (16/58) (D'Urbano, Pellegrino et al. 2010).Bos d 4 has been reported as a major allergenic protein in cow’s milk, and 51% of patients (n=92) allergic to cow’s milk have reported sensitization toward Bos d 4 (Wal 2002).

Environmental Characteristics

Source and tissue

Cow’s milk contains mainly two types of proteins, casein (Bos d 8; constitutes 80% of the total milk protein) and whey (such as Bos d 4 and Bos d 5; comprises approximately 20% of the total milk protein) (Flom and Sicherer 2019).

Bos d 4 is synthesized in the mammary gland, expressed exclusively during lactation, and is one of the most commonly found proteins in most mammalian milk (Wal 2002, Fiocchi, Brozek et al. 2010, Azdad, Mejrhit et al. 2018).

Bos d 4 belongs to the lysozyme superfamily of protein and is a regulatory subunit of lactose synthase. It alters the substrate specificity of the enzyme galactosyltransferase in the mammary gland and aids in the biosynthesis of lactose via interaction with lactose synthase (Fiocchi, Brozek et al. 2010).

According to the World Health Organization (WHO) and International Union of Immunological Societies (IUIS) database, Bos d 4 (ALA) has been identified and listed as a food allergen from cow’s milk (WHO/IUIS 2020).

Clinical Relevance

Disease severity

Cow milk allergy is considered one of the most common pediatric food allergies (Agyemang, Saf et al. 2019). Milk allergy can be either IgE-mediated or non-IgE-mediated (Walsh, Meyer et al. 2016). The clinical manifestations of cow milk protein allergy are reported to be of two types such as immediate (shortly after consumption) and delayed (after hours or days of consumption) responses (De Greef, Hauser et al. 2012). The immediate reactions are generally IgE-mediated and can lead to angioedema, urticaria, aggravation of atopic dermatitis (AD), vomiting, in some cases, anaphylaxis, as well as respiratory symptoms (breathing, wheezing, coughing) (De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012, Walsh, Meyer et al. 2016, Flom and Sicherer 2019). On the other hand, most delayed reactions are non-IgE mediated (Walsh, Meyer et al. 2016) and can lead to gastrointestinal symptoms (like enteropathy or proctocolitis) or skin reactions (such as AD) (De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012).

Milk whey proteins (such as Bos d 4) are heat-labile and therefore destroyed by extensive cooking and baking of milk (Wal 2002). Thus, the sIgE level to Bos d 4 may help assess the likelihood of tolerance to cooked or baked milk, and 75% of children with CMA may actually tolerate baked milk products such as muffins, waffles, cakes, and bread (Nowak-Wegrzyn, Bloom et al. 2008).

According to a study, anaphylaxis was reported in 6/15 patients after ingestion of cow’s milk. Furthermore, sIgE against Bos d 4 (18.8 ± 19.6 kU/L (median: 16, range: 0.51–54.9) was observed in these patients (Kiykim, Karakoc-Aydiner et al. 2019).

A study conducted among 950 children with clinically diagnosed AD reported that 60.1% (283/471) of children affected with AD and sensitized to cow’s milk (n=471) exhibited positive sIgE towards Bos d 4 (Lee, Yoon et al. 2013).

Molecular Aspects

Biochemistry

Bos d 4 is a globular protein (monomeric) of molecular weight 14.4 kDa, consisting of 123 amino acids. The protein contains four disulfide bonds within its molecule, which provide stability to its structure (Wal 2002, Tsabouri, Douros et al. 2014). It thereby has a stable protein structure with the ability to refold. The secondary structure of the protein is highly ordered and is stabilized by the presence of a calcium-binding high-affinity site (Tsabouri, Douros et al. 2014, Villa, Costa et al. 2018). The tertiary structure of this protein is spherical and rigid with an “elbow-Ca2+-binding loop” and 2 other structural domains, including an α-helical domain and a short β-sheet domain at N-terminal and C-terminal, respectively. The β-sheet domain is adjacent to the calcium-binding loop (Villa, Costa et al. 2018) and comprises 79-88 amino acid residues (Hochwallner, Schulmeister et al. 2014). In addition, cysteine residues are present on both sides of the loop, which creates four disulfide bridges, thus closing the loop and helping to stabilize the Bos d 4 structure (Hochwallner, Schulmeister et al. 2014). This protein is responsible for regulating the enzyme system of galactosyltransferase, which is involved in the biosynthesis of lactose (Wal 2002, Tsabouri, Douros et al. 2014).

Isoforms, epitopes, antibodies

According to a study, the conformation of the allergenic loops in Bos d 4 and Bos d 5 are found to have similarities, indicating the presence of characteristic conformation in their allergenic sites. The loops are distinct and firmly held as strings at both ends by antiparallel strands (Sharma, Kumar et al. 2001). The structure of sequential epitopes recognized by IgE antibodies to Bos d 4 indicated the presence of 4 IgE-binding regions (El-Agamy 2007). Likewise, in another study on 11 persistent CMA patients (4-18 years), 4 IgE-binding and 3 immunoglobulin G binding sites on Bos d 4 were identified. The IgE binding epitopes on Bos d 4 are mostly conformational rather than sequential (Järvinen, Chatchatee et al. 2001).

The epitopes are mostly present at the N- and C-terminals of the protein. The IgE epitopes remain in clusters, which may lead to cross-linking of IgE antibodies efficiently on effector cells and affect the protein's allergenic potential (Hochwallner, Schulmeister et al. 2014).

Both Bos d 4 and Bos d 5 are heat-sensitive proteins, but Bos d 4 is found to be more stable than Bos d 5 based on the molecular structure. The antigenic potential of Bos d 4 has been reported to enhance progressively with the rise in temperature from 50o to 90oC; however, a sharp reduction occurs with a further increase in temperature from 90o to 120oC. Moreover, it has been observed that heating of Bos d 4 at 120oC for 20 min lowers its allergenicity below the baseline value (Bu, Luo et al. 2009). A Moroccan study of 557 CMA patients revealed an average reduction of the allergenic potential of Bos d 4 (lowering of IgE binding) on heating at 90oC and pepsin treatment at 37oC for 1 hour by 59% and 74%, respectively (Azdad, Mejrhit et al. 2018).

Bos d 4 and Bos d 5 are reported to be heat-labile (destabilization of conformational epitopes occurs due to extensive heating). Moreover, clinical implications of Bos d 4 and Bos d 5 have been reported in patients; thus, patients reactive towards only Bos d 4 and/or Bos d 5 might be able to tolerate foods containing milk that has been extensively cooked or baked (Nowak-Wegrzyn, Bloom et al. 2008, Villa, Costa et al. 2018).

To date (7th June 2022), only one isoallergen of Bos d 4, i.e., Bos d 4.0101, has been identified and officially published by the WHO/IUIS Allergen Nomenclature Subcommittee (WHO/IUIS 2020).

Cross-reactivity

Milk of different ruminant species (such as cow, goat, and sheep) is composed of homologous proteins with similar structures and functions, thereby leading to cross-reactivity between the milk proteins (Villa, Costa et al. 2018).

A prospective cohort study conducted on 38 children (age <14 years) with CMA-associated symptoms detected the rate of cross-sensitization (skin-prick test-based) between goat milk and cow milk to be 63.2% (24/38) (Ehlayel, Bener et al. 2011).

A study reported that IgEs present in the sera of CMA children are able to recognize other mammalian (goat, sheep, and buffalo) milk proteins. However, cross-reactivity was found to be weak with milk proteins of donkeys and mares, and almost none with camel milk protein (Restani, Gaiaschi et al. 1999, El-Agamy 2007).

A study conducted among 20 patients with CMA demonstrated cross-reactivity between Bos d 4 present in cow and human milk. The results signified a greater amino acid sequence similarity between these two proteins (Maynard, Chatel et al. 1999).

Antibodies against bovine Bos d 5 have been found to exhibit cross-reactivity with the Bos d 4 present in cow’s milk. This cross-reactivity is reported due to the shared amino acid sequence between these two proteins (Baroglio, Giuffrida et al. 1998).

Bos d 4 from cow’s milk protein shows sequence homology with water buffalo (99.3%), ewe (97.2%), goat (95.1%), pig (74.6%), donkey (71.5%), horse (72.4%), and camel (69.7%) (Tsabouri, Douros et al. 2014).

Diagnostic Relevance

Exposure

Cow milk allergy mainly occurs through ingestion (Kaczmarski, Wasilewska et al. 2013).

Compiled By

Author: Turacoz Healthcare Solutions

Reviewer: Dr. Eva Södergren

Explained Results

Allergen Information

Bos d 4 (α-lactalbumin) is considered a major allergenic protein in cow’s milk (Villa, Costa et al. 2018).

Clinical relevance

Typical symptoms of CMA include skin reactions (angioedema, urticaria, and atopic dermatitis) (De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012, Lee, Yoon et al. 2013, Flom and Sicherer 2019), gastrointestinal symptoms (De Greef, Hauser et al. 2012), respiratory systems, and in some cases, anaphylaxis (Kiykim, Karakoc-Aydiner et al. 2019).

Cross-reactivity

Antibodies against bovine Bos d 4 show cross-reactivity with Bos d 5 present in cow’s milk due to shared amino acid sequence (Baroglio, Giuffrida et al. 1998). Bos d 4 from cow’s milk shows sequence homology with other mammalian milk proteins, including water buffalo, ewe, goat, pig, donkey, horse, and camel (Tsabouri, Douros et al. 2014). Furthermore, cross-reactivity has also been reported between Bos d 4 in cow and human milk (Maynard, Chatel et al. 1999).