Galacto-oligosaccharides (GOS) are prebiotics, which are used widely supplemented in commercial infant formula and beverages for their ability to selectively promote the growth of beneficial gut bacteria in the human intestine, such as bifidobacteria (1). GOS is a complex mixture consisting of oligosaccharides with 1 up to 7 galactose units linked to a glucose terminal. The degree of polymerization (DP) of an oligosaccharide reflects the number of sugar units (2). GOS synthesized commercially from β-galactosidase enzyme derived from Bacillus circulans consists mainly of β-1,4-linked GOS. Other β-glycosidic linkages between the monomer units such as 1-6 Gal, 1-2 Glc, 1-3 Glc, 1-4 Glc, 1-6 Glc, 1-2 Gal, and 1-3 Gal also exist and can form linear and branched linkages (3). The structures and graphical representation of the permutations of linkages for DP2, DP3, and DP4 are depicted in Table 1.

Soon after the introduction of GOS supplemented infant and maternal milk formula to South East Asia, cases of IgE-mediated immediate allergic reactions and anaphylaxis linked to GOS were reported (4, 5). These reactions occurred upon first consumption of these products and the putative primary sensitizer has been determined to be Blomia tropicalis (Blo t) (6).

GOS is a unique allergen as it is a pure carbohydrate. The conventional allergen is a protein or glycoprotein that is capable to initiate T cell responses and bind allergen specific IgE. Even though a vast diversity of anti-glycan IgE is detectable in circulation, the binding of this IgE to cross-reactive carbohydrate determinants have been shown to be weak in affinity and, therefore, clinically irrelevant (7). This concept was challenged with the description of allergy to alpha-gal, where IgE to this carbohydrate epitope resulted in Cetuximab-induced anaphylaxis (8).

In contrast to α-gal, an epitope that lies within a glycoprotein through N-glycosylation (9); GOS is a more unusual allergen as it is a pure carbohydrate with an average molecular weight of <1,000 daltons. For effective activation of basophils, allergens require at least two IgE epitopes for cross-linking of IgE (10). It is still an unresolved question how a small carbohydrate like GOS cross-links IgE. This study aimed to evaluate the possible mechanisms by which GOS activates basophils; specifically, we determined if cell surface proteins on basophils and/or other immune cells act as carrier proteins to cross-link IgE. Galectins on cell surfaces were selected as a plausible candidate, as they are a class of lectins that bind specifically to β-galactoside carbohydrates via a carbohydrate recognition domain (11). In this study, we show that cross-linking of IgE on basophils by GOS does not require the binding of GOS to galectins or to other cell surface proteins. We speculate that GOS itself is, therefore, likely to be able to cross-link IgE on basophils.

GOS is a mixture of oligosaccharides consisting of variable degrees of polymerization of galactose units (up to 7) linked to a glucose terminal (21). In our previous description of GOS allergy, we showed that basophil activation by GOS required a minimum of three sugar moieties (i.e. DP3 with 2 galactose and 1 glucose) (4). It remains a puzzle as to how these oligosaccharides of low molecular weight (~540 daltons) could cross-link IgE on basophils which by criteria requires at least two IgE binding epitopes in order to bind two molecules of cell surface IgE simultaneously. In this study, we examined possible mechanism(s) by which GOS as an allergen activates basophils.

Our first approach was to consider the possible role of lectins that may participate in GOS-specific IgE cross-linking on basophils. Basophils are shown to have surface expression of some C-type lectins such as CLECSF14, DEC205, Dectin-1, Dectin-2, and mannose receptor C 2 (MRC2) and I-type lectins, immunoglobulin-like lectin (Siglec)-8 (22, 23). However, none of these lectins are known to bind β-galactoside sugars. On the other hand, basophils also express galectins which are a family of animal lectins that bind β-galactoside containing sugars with high specificity. As many as 12 galectins have been found in humans (24). They are present as cytoplasmic or secreted forms. The cytoplasmic form is usually monomeric and the secretory form can become di- or pentameric. After their secretion, they can bind to O- or N-linked glycans of cell surface proteins, such as CD43, CD45, TCR, Tim-3, Fc epsilon receptor I (25, 26). Galectins-1,−3,−8, and−9 are commonly bounded to the surface molecules of immune cells. Furthermore, galectins may trigger receptor cross-linking due to their di- or multi-valent characteristics. Since GOS is composed of different galactosyl residues (21), it is likely that GOS binds to galectins. Our earlier studies showed that basophils could be activated by GOS in serum-free conditions, indicating that circulating plasma factors such as soluble galectins are not likely to participate in GOS-mediated basophil activation (4). It is, however, possible that membrane-bound galectins acts as carrier molecules for GOS to cross-link IgE. Galectin-3 in particular can undergo conformational change to form pentamers upon ligand binding. GOS may, therefore, become multivalent through binding to pentameric galectin-3.

In this study, we examined the involvement of galectins in GOS-mediated basophil activation in whole blood assay using five different galectin inhibitors. Our results, however, showed that three of the five high affinity galectin inhibitors TD139, GB1107, and G3-C12 are used, failed to inhibit GOS-triggered basophil activation (Figure 1B), hence, ruling out the involvement of galectins-1 and−3 in the GOS-mediated basophil activation.

Contrasting results were obtained with the remaining two galectin inhibitors, TDG and lactose. Both showed inhibitory effects on GOS-mediated basophil activation at low concentrations of GOS, but this inhibition was diminished/lost at higher GOS concentrations (Figure 1B). Since both TDG and lactose have lower affinity for galectins compared to the former three inhibitors, we postulated that these observed inhibitory effects on GOS-mediated basophil activation were not likely due to galectin inhibition per se.

In view of the structural similarities between GOS, lactose, and TDG, we explored the possibility that these galectin inhibitors inhibit the binding of GOS to GOS-specific IgE. Indeed, the GOS IgE ELISA inhibition showed that both lactose and TDG inhibited the GOS IgE binding in a dose-dependent manner, with inhibition up to 2–3 log concentrations higher for lactose and TDG than self-inhibition with GOS and enriched GOS fraction (DP4) (Figure 2).

We next examined the possibility that GOS may obtain di- or multi-valent IgE binding capacity through binding with other types of lectins on immune cell surface molecules. Basophil activation tests using GOS, with anti-IgE and Blo t controls, were performed using purified basophils. A reduction in basophil activation was observed with all three stimulants, with the highest reduction for GOS (two samples negative), followed by anti-IgE (1 sample negative), and Blo t showed the least reduction (Figure 3). The two subjects whose purified basophils showed negative BAT results to GOS also showed >50% reduction in the percentage of CD63⁺ cells (one subject negative) to control stimulant anti-IgE. We, therefore, speculate that the purification steps reduced the density of IgE molecules on basophils and may increase the threshold for basophil activation, hence, affecting the responses to anti-IgE and GOS stimulation. These effects were less marked with Blo t stimulation, and a plausible reason is that Blo t allergens are multivalent whereas anti-IgE is divalent. The number of IgE epitopes per GOS is still unknown but it is likely fewer than Blo t in view of its molecular weight. Furthermore, for BAT using whole blood samples, basophil activation was achieved with the concentrations of Blo t were 10–100 times less than GOS (Figure 1A) indicating that Blo t may have a higher affinity to IgE than GOS and hence the degree of basophil activation is less affected by this increased threshold. Taken together, despite the loss of basophil activation in two subjects, our data suggest that GOS is likely to be able to cross-link IgE on basophils independently.

Although the IgE binding epitopes of GOS allergy are still unresolved, our competitive ELISA assay indicates that disaccharides (lactose and TDG) can bind GOS-specific IgE. Of note, lactose and TDG are disaccharides composed of different sugar molecules suggesting that GOS-specific IgE is not monoclonal, with higher affinity for the structure with two galactose rings (TDG) than one galactose plus one glucose (lactose).

We, therefore, speculate that at least some oligosaccharides in the mixture of GOS are likely to possess two or more IgE epitopes. However, it is known that binding of two antibodies (such as IgE) to epitopes in close proximity can be blocked by steric hindrance, especially for small molecules (27). In this regard, Kaneko et al. have shown that a GOS linear tetrasaccharide with only β1-4 linkage is unable to activate basophils but the linear structure Galβ1-4Galβ1-4Galβ1-3Glc and branched structure Galβ(1-4)Galβ(1-6)-[Galβ(1-4)]-Glc (Table 1 in bold) are allergenic in GOS allergy subjects in Japan (28). For this latter structure, we have previously confirmed that it does not trigger the activation in basophils from Singapore GOS allergic subjects, suggesting that GOS allergy can be caused by different GOS structures in different regions (29). Although the exact allergenic GOS structures responsible for GOS allergy in Singapore are still unknown, our study also indicated that the enriched DP4 fraction with more branched GOS structures from specifically produced GOS is more potent than DP4 fraction from commercial vGOS in inducing basophil activation in vitro (6). Branched GOS structures may be more likely to overcome the steric hindrance and cross-link IgE on basophils.

In conclusion, we have demonstrated that GOS-mediated IgE cross-linking does not require galectin as carrier. GOS may be able to cross-link IgE independently.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Review Board of the National Healthcare Group, Singapore. Written informed consent to participate in this study was provided by participants/participants' legal guardian.

LL conducted experiments and performed data analysis. SL conducted experiments, performed data analysis, and drafted manuscript. HW conducted experiments. JS, WC, YZ, ET, and WL contributed to the sample collection, data interpretation, and revised the manuscript. DD provided the materials required for the study and contributed to the data interpretation and revised the manuscript. BL contributed to the study design, data interpretation, and revised the manuscript critically. C-HH conceived the study and study protocol and participated in manuscript writing. All authors contributed to the article and approved the submitted version.

This work was supported by the National Medical Research Council, Singapore (NMRC/CIRG/1487/2018).

DD is employed by FrieslandCampina, Amersfoort, The Netherlands. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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