Lectin microarray profiling and monosaccharide analysis of bovine milk immunoglobulin G oligosaccharides during the first 10 days of lactation

Abstract

Immunoglobulin G (IgG) in bovine milk is credited with ensuring efficient passive immunity for newborn calves. Bovine milk IgG glycosylation may also have positive impacts on the health of nonbovine consumers of cow's milk. Milk IgG's glycosylation contributes to effector function and may also protect it from protease digestion, allowing IgG to reach the intestine for absorption. However, relatively little is known about changes in milk IgG oligosaccharide presentation and composition over early lactation. In this work, IgG was isolated from milk pooled from three cows at four time points over the first 10 days of lactation postparturition. Purified IgG was labeled with a fluorescent dye and interrogated with a microarray consisting of 48 carbohydrate-binding proteins (lectins) from plant, fungal, and bacterial sources. Lectin microarray profiles suggested that only subtle changes in the glycosylation of IgG occurred during days 2 and 3 of lactation, but by day 10, the lectin profile diverged from the other three time points. Monosaccharide analysis carried out after hydrolysis confirmed that the ratios of oligosaccharide components remained relatively stable through day 3 and also that sialylation was substantially reduced by day 10. The differences that were observed for glycosylation suggest that different functionalities associated with IgG glycosylation may be required in the first days of life.

Keywords: glycosylation; immunoglobulin; lactation; lectin; microarray; milk.

Figure 1

Purification of IgG from bovine milk. (a) Composite comparison of peaks from individual elution chromatograms showing the relative decrease in quantity of IgG obtained from equal loading of milk from each day. (b) Composite image of Coomassie‐stained SDS‐PAGE gels showing that the purity of the isolated IgG fractions (days 1, 2, 3, and 10) against the purchased IgG standard (Std). Lane MW is the molecular weight standard

Figure 2

Comparison of the normalized profiles for IgG isolated from milk at days 1, 2, 3, and 10 postparturition. (a) Set one at 0.5 μg/ml loading and (b) set two at 1.0 μg/ml loading. Responses are averages for replicates with error bars representing ±1 SD

Figure 3

Unsupervised hierarchical clustering of the normalized lectin microarray responses for IgG from days 1, 2, 3, and 10 pooled milk samples (totaling 36 replicates, “B” indicates additional technical replicates of each sample compared on one single microarray). All data subjected to total intensity mean normalization prior to 2‐dimensional clustering using the average linkage, Euclidean distance method. Blue box at right indicates the cluster of day 10 profiles (6 out of 8 replicates) which demonstrated 56% minimum similarity to the balance of the samples with this clustering method. Day 1, n = 10; Day 2, n = 10; Day 3, n = 8; Day 10, n = 8). “Empty” represents the unprinted microarray slide surface, “PBS” buffer only, and “BSA” is bovine serum albumin