Glycoprotein expression in human milk during lactation

Abstract

While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

Figure 1

Coomassie (top) or glycoprotein-specific (bottom) stained SDS-PAGE separations for 3 mothers at varying time points. Indicated protein bands identified by peptide mass fingerprinting are annotated in Table 1 below, along with several measures of the quality of the identification. Abbreviations are: M, mother code; D, lactation day; MWM, molecular weight markers. Markers for coomassie stain are, from top, 203, 120, 95, and 49 kDa. For pro-Q stain, markers are 180, 82, and 42 kDa. Protein K is alpha-lactalbumin.

Figure 2

Electropherograms of SDS-PAGE separated, coomassie stained proteins with annotated identifications as detailed in Table 1. Protein K is alpha-lactalbumin.

Figure 3

Glycosylation (upper) or expression (lower trace) levels of selected human milk glycoproteins. Singly, doubly, or triply marked entries are statistically different at the 90, 95, or 99% confidence interval from the corresponding day 1 entry. Standard deviations for each measurement are shown as error bars. When necessary, signals were scaled by the factor in parentheses to enable direct comparison between highly and moderately abundant glycoproteins.

Figure 4

Image line profiles of isolated milk proteins stained with either Coomassie (left) or Pro-Q (right) stains. Proteins were isolated from a single mother on lactation days 1, 2, 5, and 10. While lactoferrin is shown to have a relatively constant total protein level (top left), the degree of glycosylation at day 10 is approximately 40% of that present at day 1 (top right). The lower two traces showing Ig α-1 chain C behavior show little variation.

Figure 5

Variations of lactoferrin glycosylation during lactation. Representative mass spectra of enzymatically released lactoferrin N-glycans from lactation days 2 (top) and 5 (bottom) from a single individual are shown. Monosaccharide compositions of the major peaks are annotated as H, Hexose; N, N-acetyl hexosamine; and F, deoxyhexose. A dramatic decrease in both quantity and diversity of glycosylation is observed between lactation days 2 and 5, as shown by the glycosylation-specific Pro-Q staining.