In Depth Analysis of the Contribution of Specific Glycoproteins to the Overall Bovine Whey N-Linked Glycoprofile

Abstract

The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely, lactoferrin, lactoperoxidase, α-lactalbumin, immunoglobulin-G (IgG), and glycosylation-dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (∼95%) of N-glycans present in the overall whey glycoprofile were attributed to three proteins: lactoferrin, IgG, and GlyCAM-1. We identified specific signature glycans for these main proteins; lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-β(1,4)-GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin, and GlyCAM-1 over time.

Keywords: GlyCAM-1; PP3; colostrum; milk; protein glycosylation; whey.

Figure 1

Overall acid whey HPLC glycoprofile obtained for pooled milk from Holstein-Friesian cows. Glycan structures detected and identified by MS were added to the spectrum; for a full overview of all structures, see Table S2 of the Supporting Information. Structures with main contributions to the peak intensity are marked with an asterisk.

Figure 2

Overlay of the neutral (black line) and acidic (sialylated, red line) glycan fractions of the overall whey HPLC glycoprofile, obtained from pooled milk of Holstein-Friesian cows. The 2-AB-labeled glycans were divided into neutral and acidic fractions by anion-exchange chromatography fractionation.

Figure 3

HPLC chromatograms of the glycan fingerprints of individual glycoproteins present in bovine acid whey. Glycan structures of the most abundant peaks were annotated.

Figure 4

HPLC chromatograms of the glycan fingerprints of the individual whey glycoproteins shown at their reported physiological concentrations. (A) Overall whey glycoprofile is shown at the top (undiluted), and (B) individual glycan fingerprints are shown at the bottom. The proteins were overlaid in the following concentration ratios: heated whey (undiluted) (GlyCAM-1), lactoferrin (0.20 mg/mL), lactoperoxidase (0.05 mg/mL), IgG (0.40 mg/mL), and α-lactalbumin (1.5 mg/mL). A selection of structures of the individual proteins was annotated; for the full annotation, refer to Figure 1. The main glycan of lactoferrin, Man-9, is shown as a starred structure.

Figure 5

HPLC chromatograms of whey glycoprofiles of 8 times diluted colostrum (bottom) and undiluted (undil.) colostrum versus mature milk (top). Glycoprofiles displayed are from (left) cow 1 and (right) cow 3 (Table S1 of the Supporting Information). A selection of structures from lactoferrin (LF), immunoglobulin G (IgG), and GlyCAM-1 (GCM) was annotated. Sections with multiple co-eluting tri-antennary (Tri) and tetra-antennary (Tetra) glycans are bracketed. Additional glycoprofiles from cows 2, 4, 6, 7, and 8 are provided in Figures S5–S10 of the Supporting Information.