Dynamic temporal variations in bovine lactoferrin glycan structures

It has been reported previously that glycosylation of bovine lactoferrin changes over time. A detailed structural overview of these changes over the whole course of lactation, including predry period milk, is lacking. In this study, a high-throughput analysis method was applied to the glycoprofile of lactoferrin isolated from colostrum, mature, and predry period mature milk, which was analyzed over two subsequent lactation cycles for 8 cows from diverse genetic backgrounds. In addition, comparisons are made with commercial bovine lactoferrin samples. During the first 72 h, dynamic changes in lactoferrin glycosylation occurred. Shifts in the oligomannose distribution and the number of sialylated and fucosylated glycans were observed. In some cows, we observed (α2,3)-linked sialic acid in the earliest colostrum samples. The glycoprofiles appeared stable from 1 month after delivery, as well as between cows. In addition, the glycosylation profiles of commercial lactoferrins isolated from pooled mature milk were stable over the year. Lactoferrin glycosylation in the predry period resembles colostrum lactoferrin. The variations in lactoferrin glycosylation profiles, lactoferrin concentrations, and other milk parameters provide detailed information that potentially assists in unraveling the functions and biosynthesis regulation of lactoferrin glycosylation

Quantitative analysis of bovine whey glycoproteins using the overall N-linked whey glycoprofile

Bovine whey is an important ingredient in human nutrition and contains many biofunctional, glycosylated proteins. Knowledge on the glycoprotein composition of whey and whey products is valuable for the dairy industry. This paper describes a method for the characterisation of whey, or whey powders, by N-linked glycoprofile analysis. Application of the method for analysis of whey protein products showed clear differences in glycoprotein composition between concentrate, isolate and demineralised whey powders. The quantitative potential was explored by screening 100 pooled farm milk samples. IgG and lactoferrin protein concentrations determined by N-glycoprofile analysis matched well with ELISA results. The protein concentration of GlyCAM-1 was determined to be ≥1 mg mL−1. The approaches presented in this work allow simultaneous concentration estimation of the three major whey glycoproteins, lactoferrin, IgG and GlyCAM-1 on the basis of their N-linked glycoprofiles, also in highly processed samples where conventional methods of detection (ELISA) are less suitable

In depth analysis of the contribution of specific glycoproteins to the overall bovine whey N-linked glycoprofile

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