Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions

Abstract

Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.

Fig. 1.

N-Glycoprofile of commercial human milk Lactoferrin. A, Positive ion mode MALDI-FTICR mass spectrum of N-glycans recovered in 20% AcN:water fraction. B, Positive ion mode MALDI-FTICR mass spectrum of N-glycans recovered in 40% acetonitrile/water fraction, and C, Negative ion mode MALDI-FTICR mass spectrum of N-glycans recovered in 40% AcN:water.

Fig. 2.

Degree of glycosylation of human milk LF from individual donors across lactation. All glycan signals were summed and the abudances normalized relative to Day 1, which was regarded as 100%. Unfortunately, mother 4 did not produce milk on Day 1 and therefore was normalized to Day 5. A paired t test was performed to determine statistically significant differences between Day 1 and other days.

Fig. 3.

Changes in bacterial adhesion with hmLF addition and the purified glycan from hmLF. Bars below the x axis indicate the p value between the respective treatments. Control is adhesion of the respective bacterium without glycan addition. Bacterial isolates used were ST = Salmonella enterica ssp enterica typhimurium; SE = Salmonella enterica ssp enterica enteritidis; SH = Salmonella enterica ssp enterica heidelberg; LM = Listeria monocytogenes; EC = Escherichia coli O157:H7.

Fig. 4.

Bacterial association changes with modification of the hmLF glycosylation. Bacteria were incubated with CaCO-2 cells in the presence of hmLF (hLF), hmLF treated with sialidase (Sialic acid free), hmLF treated with fucosidase (Fucose free), hmLF treated with sialidase and fucosidase (Sia and Fucose free) hmLF treated with galactosidase (Galactose free) and N-glycans released from hmLF (N-glycans). Baseline infection (infection) was determined without any additives. Gray bars above the horizontal line represent the number of bacteria adhered to the epithelial surface during 60 min of incubation, whereas black bars below the horizontal line represent the amount of bacteria invaded into the epithelial cell during the same incubation period.