f77 nBos d 5, β-lactoglobulin 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 5 (β-lactoglobulin) has been reported as a major allergenic protein in cow's milk. Sensitization to Bos d 5 has been reported to range between 13-76% in cow’s milk allergy patients. Milk whey proteins (like Bos d 5) are heat-labile and therefore are likely to be destroyed after extensive cooking and baking of milk. Thus, specific immunoglobulin E (sIgE) to Bos d 5 might help assess the tolerance to cooked or baked milk. Further, a lower level of sIgE against Bos d 5 has been reported to indicate tolerance towards cow’s milk. Clinical manifestations of Bos d 5-mediated allergy majorly include anaphylaxis and atopic dermatitis. Moreover, sIgE testing with Bos d 5 has been considered vital in diagnosing a cow’s milk protein allergy. Cross-reactivity has been observed between antibodies against bovine Bos d 5 and α-lactalbumin in cow's milk due to the shared amino acid sequence. Moreover, Bos d 5 from cow's milk has been found to exhibit sequence homology with other mammalian milk proteins (like water buffalo, ewe, goat, pig, donkey, and horse).

Epidemiology

Worldwide distribution

Cow's milk is popularly consumed throughout the world (Muthukumar et al. 2020). However, 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 (Flom et al. 2019, Skripak et al. 2007). Bos d 5 (β-lactoglobulin; BLG) has been reported as a major allergenic protein in cow's milk. Sensitization to Bos d 5 has been reported to range between 13-76% in cow’s milk allergy patients (Fiocchi et al. 2010).

According to a study, 61% of patients (n=92) allergic to cow's milk have reported sensitization toward Bos d 5 (Wal 2002).

A Taiwan-based retrospective study reported sensitization towards Bos d 5 among 46.8% (89/190) of individuals with specific immunoglobulin E (sIgE) towards whole cow's milk (Chen et al. 2014). Furthermore, a study conducted in Poland among 291 children (2-14 years) with a history of cow's milk allergy (CMA) reported sensitization towards Bos d 5 in 53.6% (37/69) of children (Kaczmarski et al. 2013).

According to a study conducted in Japan on 83 children (0.8-15.8 years) with suspected CMA, the clinically diagnosed CMA children (n=61) were reported to possess considerably elevated levels of Bos d 5 sIgE compared to the non-CMA children (Ito et al. 2012). In addition, allergy against Bos d 5 was reported in 63.4% of patients with CMA (n=86) in an Iran-based study (Shokouhi Shoormasti et al. 2016).

A Netherland-based study included 30 (≥16 years) patients with CMA [18 assessed via skin prick test (SPT) and 10 via double-blind placebo-controlled food challenge (DBPCFC)] in the study group and 25 individuals in the control group (cow milk sensitized but tolerant individuals). The study reported significantly higher reactivity (through SPT) towards Bos d 5 (p=0.004) among patients in the study group compared to the control group. Furthermore, sIgE towards Bos d 5 was observed in 56% (10/18) of CMA patients (Lam et al. 2008).

A study in Germany and Denmark used crossed radioimmunoelectrophoresis (CRIE) technique to determine the level of serum IgE to cow milk antigenic components in the sera of 21 CMA patients and detected IgE antibodies against Bos d 5 in the sera of all patients (Gjesing et al. 1986).

Environmental Characteristics

Source and tissue

Cow's milk contains mainly two types of proteins that include casein (80% of the total milk protein) and whey (20% of the total milk protein) (Flom et al. 2019). Bos d 5 (whey protein) is abundantly available in cow's milk (Fiocchi et al. 2010), synthesized in the epithelial cells of the mammary gland (Broersen 2020, Wal 2002). It is also found in the milk of other mammals; however, it is absent in human milk (Fiocchi et al. 2010).

Bos d 5 is a globular protein (lipocalin superfamily) (Fiocchi et al. 2010, Villa et al. 2018) and has a strong binding affinity for small hydrophobic ligands such as cholesterol, fatty acids, retinol, etc. (Villa et al. 2018). In addition, this protein is anticipated to be responsible for mediating cellular processes via ligand binding (Roth-Walter et al. 2014).

According to the WHO/IUIS database, Bos d 5 has is listed as a food allergen from cow's milk (WHO/IUIS 2020).

Clinical Relevance

Cow milk allergy is regarded as a major common pediatric food allergy (Agyemang et al. 2019). Milk allergy can be either IgE-mediated or non-IgE-mediated (Walsh et al. 2016). The clinical manifestations of cow's 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 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 et al. 2012, Flom et al. 2019, Ito et al. 2012, Walsh et al. 2016). On the other hand, most delayed reactions are non-IgE mediated (Walsh et al. 2016) and can lead to gastrointestinal symptoms (like enteropathy or proctocolitis) or skin reactions (such as AD) (De Greef et al. 2012, Ito et al. 2012).

Milk whey proteins such as Bos d 5 are heat-labile and therefore destroyed during the extensive cooking and baking of milk (Wal 2002). Thus, the sIgE level to Bos d 5 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 (Anna Nowak-Wegrzyn 2016, Caubet et al. 2013, Ford et al. 2013). High sIgE levels of Bos d 5 indicate an allergy to all forms of milk (Caubet et al. 2013, Ford et al. 2013), and low sIgE levels of Bos d 5 may indicate the development of tolerance (Sicherer et al. 1999).

According to a study, anaphylaxis was reported in 6/15 patients after ingestion of cow's milk. Furthermore, sIgE against Bos d 5 (15.3 ± 16.2 kU/L; median: 7.2, range: 0.14–53) was observed in these patients (Kiykim et al. 2019).

In a Korea-based study, 950 children with clinically diagnosed AD reported that 52.5% (245/471) of children affected with AD and sensitized to cow's milk (n=471) exhibited positive sIgE towards Bos d 5 (Lee et al. 2013).

Cross-reactive molecules

A prospective cohort study conducted on 38 children (age <14 years) with CMA-associated symptoms reported cross-sensitization (SPT-based) between goat and cow milk (Ehlayel et al. 2011).

Molecular Aspects

Biochemistry

Bos d 5 is a small protein of molecular weight 18.3 kDa comprising 162 amino acids (Hochwallner et al. 2014). It occurs as an equilibrium mixture of monomer and dimer forms under physiological conditions. However, the dimers may connect to octamers at their isoelectric point (Fiocchi et al. 2010). Each subunit of Bos d 5 contains amino acids that involve two disulfide bonds and one free cysteine, which leads to the dimerization of the molecule (Villa et al. 2018).

The presence of these internal disulfide bonds is responsible for the high stability of Bos d 5 against acidic hydrolysis and proteolysis, retaining its structural integrity following digestion and facilitating its absorption via the intestinal mucosa (Hochwallner et al. 2014, Villa et al. 2018). This high stability of Bos d 5 may be accountable for its high allergenic potential (Hochwallner et al. 2014).

Moreover, according to X-ray crystallization and nuclear magnetic resonance technique, the tertiary structure of Bos d 5 has been claimed to be globular in shape. Further, it is built on an 8-stranded antiparallel beta-barrel with a 3-turn alpha-helix on the outer surface and the ninth β-strand adjoining the first strand (Hochwallner et al. 2014); this ninth β-strand is responsible for dimer formation (Broersen 2020, Wal 2002).

Isoforms, epitopes, antibodies

Bos d 5 has been found to possess two principal isoforms that include genetic variants A (BLGA) and B (BLGB), which only vary due to two-point mutations in amino acids at positions 64 and 118 (aspartic acid and valine in BLGA and glycine and alanine for BLGB) (Fiocchi et al. 2010, Hochwallner et al. 2014). This protein contains several IgE-binding linear epitopes (Linhart et al. 2019, Villa et al. 2018), of which 7 IgE-binding epitopes have been identified on Bos d 5 (El-Agamy 2007).

The heating of milk above 60oC may destabilize β-sheets and unfold the β-barrel leading to the exposure of free cysteine and disulfide bonds in the solvent. Further heating of milk above 65oC causes irreversible protein denaturation due to new hydrophobic interaction and the loss of secondary and tertiary protein structures. This reorganization of the Bos d 5 protein structure may mask the sequential or linear epitopes, which may account for the development of tolerance towards boiled milk rather than raw milk in some CMA patients (Tsabouri et al. 2014).

To date (31st May 2022), only one isoallergen of Bos d 5, i.e. Bos d 5.0101, has been identified and officially published by the WHO/IUIS (WHO/IUIS 2020).

Cross-reactivity

Cow, goat, and sheep milk consists of homologous proteins with similar structures and functions, thereby leading to cross-reactivity between the milk proteins (Villa et al. 2018).

Bos d 5 from cow's milk protein shows sequence homology with other mammalian milk, including water buffalo (96.7%), ewe (93.9%), goat (94.4%), pig (63.9%), donkey (56.9%), and horse (59.4%) (Tsabouri et al. 2014).

Additionally, antibodies against bovine Bos d 5 have been reported to exhibit cross-reactivity with ALA both in the native and denatured state (Baroglio et al. 1998, El-Agamy 2007). This cross-reactivity is reported to occur due to the shared amino acid sequence between these two proteins (Baroglio et al. 1998).

Diagnostic Relevance

Disease Severity

Bos d 5-sIgE has been considered essential in diagnosing cow milk protein allergy (El-Sebay et al. 2015). According to a study, the level of sIgE to Bos d 5 and Bos d 5 sIgE/IgG4 ratios were significantly elevated in patients who were reactive to baked milk compared to patients who were tolerant to baked milk (Caubet et al. 2013).

According to a study, the level of sIgE against Bos d 5 has been anticipated to be associated with immediate reactions, family history and the eosinophil percentage (El-Sebay et al. 2015).

The presence of several IgE-binding linear epitopes on Bos d 5 may suggest the significance of its particular peptides to be used as molecular markers to diagnose persistent CMA (Villa et al. 2018).

Exposure

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

Compiled By

Author: Turacoz Healthcare Solutions

Reviewer: Dr. Eva Södergren

Explained Results

Allergen Information

Bos d 5 (β-lactoglobulin) is considered a major allergenic protein in cow's milk (Wal 2002).

Clinical information

Characteristic symptoms of CMA include skin reactions (angioedema, urticaria, and atopic dermatitis) (De Greef et al. 2012, Flom et al. 2019, Ito et al. 2012, Lee et al. 2013), gastrointestinal symptoms (De Greef et al. 2012), respiratory systems, and in some cases, anaphylaxis (Kiykim et al. 2019).

Cross-reactivity

Antibodies against bovine Bos d 5 show cross-reactivity with ALA present in cow's milk due to shared amino acid sequence (Baroglio et al. 1998). In addition, Bos d 5 in cow's milk shows sequence homology with other mammalian milk proteins, including water buffalo, ewe, goat, pig, donkey, and horse (Tsabouri et al. 2014).