Human milk oligosaccharides and Lewis blood group: individual high-throughput sample profiling to enhance conclusions from functional studies

Abstract

Human milk oligosaccharides (HMO) are discussed to play a crucial role in an infant's development. Lewis blood group epitopes, in particular, seem to remarkably contribute to the beneficial effects of HMO. In this regard, large-scale functional human studies could provide evidence of the variety of results from in vitro investigations, although increasing the amount and complexity of sample and data handling. Therefore, reliable screening approaches are needed. To predict the oligosaccharide pattern in milk, the routine serological Lewis blood group typing of blood samples can be applied due to the close relationship between the biosynthesis of HMO and the Lewis antigens on erythrocytes. However, the actual HMO profile of the individual samples does not necessarily correspond to the serological determinations. This review demonstrates the capabilities of merging the traditional serological Lewis blood group typing with the additional information provided by the comprehensive elucidation of individual HMO patterns by means of state-of-the-art analytics. Deduced from the association of the suggested HMO biosynthesis with the Lewis blood group, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiles of oligosaccharides in individual milk samples exemplify the advantages and the limitations of sample assignment to distinct groups.

Figure 1

Biosynthesis of neutral complex human milk oligosaccharides (HMO). The assumed biosynthetic pathway starts from the activated monosaccharides and includes the most important enzymes only [N-acetylglucosaminyltransferases (GlcNAcT)]: iβ3GlcNAcT attaches N-acetylglucosamine (GlcNAc) in the β1–3 position to terminal galactose (Gal), Iβ6GlcNAcT attaches GlcNAc in β1–6 position to terminal Gal. Galactosyltransferases (GalT): β3GalT attaches Gal in the β1–3 position to GlcNAc and β4GalT attaches Gal in the β1–4 position to GlcNAc. Fucosyltransferases (FucT): α2FucT attaches fucose (Fuc) in the α1–2 position to terminal Gal, secretor (Se) enzyme, α3FucT attaches Fuc in the α1–3 position to GlcNAc, α3/4FucT attaches Fuc in the α1–3/4 position to GlcNAc and in the α1–3 position to Glc of the lactose core, Lewis (Le) enzyme . The no entry signs mean that no further elongation takes place. Fucosylation is indicated exemplarily for terminal type 1 and type 2 chains. Glycan structures are depicted according to the recommendations of the Consortium of Functional Glycomics using the GlycoWorkbench software tool (94).

Figure 2

The Lewis (Le) and Secretor (Se) gene–related glycan epitopes. The Le and Se epitopes, which are characteristic for the Le phenotype in red blood cells and in human milk, are synthesized by the listed fucosyltransferases (FucTs). The Le and Se genes code for the active FucTs in presence of at least 1 functional allele (heterozygous with Lele or Sese, homozygous with LeLe or SeSe). The prevalence of the Le phenotypes is conferred to Europeans (8). Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine.

Figure 3

Matrix-assisted laser desorption/ionization time-of-flight MS profile spectra of 4 individual milk samples. Spectra displayed were obtained from 4 women: 2 serologically typed as Le(a−b−) (A,B) and 2 as Le(a−b+) (C,D), respectively. The signals represent sodium adducts. The 95% CI, calculated for each human milk oligosaccharide composition, base on data from 40 individual milk samples. CI are illustrated by open bars if the measured signal intensity is not in the expected range and by shaded bars if it is. Relative CIs are described in Reference and are applied to the measured signal intensities. Due to high variance in the signal intensities of the high molecular weight HMO, CIs were only calculated for signals up to m/z 1533. Compositions are calculated using GlycoPeakfinder software (95).

Figure 4

Matrix-assisted laser desorption/ionization time-of-flight MS/MS analysis of purified human milk oligosaccharides of a Lewis (a+b−) donor. Inset shows range from m/z 650 to m/z 720 at 50× magnification. The obtained fragment ions were assigned according to the recommendations of the Consortium of Functional Glycomics using GlycoWorkbench (94). Fragment ions are designated in accordance with the nomenclature of Domon and Costello (85). In some cases, fragments may be formed by different fragmentation pathways, only 1 of which is illustrated. All fragment ions represent sodium adducts. The unexpected signal is circled in red.

Figure 5

Discriminant analysis. The results obtained for 113 single matrix-assisted laser desorption/ionization time-of-flight MS and redundant MS/MS measurements of 40 milk samples underwent discriminant analysis. Discriminant function 1 is plotted on the x-axis and discriminant function 2 on the y-axis. Open diamonds, red squares, and green triangles represent HMO samples from Le(a−b+), Le(a+b−), and Le(a−b−) donors, respectively. The distribution of each group is indicated by colored shading. Reproduced with kind permission from Springer Science+Business Media (74), Figure 7.