Dynamic Temporal Variations in Bovine Lactoferrin Glycan Structures

Abstract

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 highthroughput 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.

Variations in N-linked glycosylation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) whey protein: Intercow differences and dietary effects

“…Each of these major glycoproteins expresses a unique glycan fingerprint. In previous work we structurally identified all glycans represented in the peak clusters of these glycoproteins (Valk-Weeber et al, 2020b). However, glycan structures of similar length and complexity coelute in 1 peak cluster, and some glycans may originate from more than 1 protein.…”

“…Glycans were released by peptide-N-glycosidase F treatment (100 units/ sample; PNGase F, Flavobacterium meningosepticum, New England Biolabs, Ipswich, UK). Glycan labeling via reductive amination and cleanup was performed as described by Ruhaak et al (2008) and Valk-Weeber et al (2020b), with anthranilic acid (Sigma-Aldrich) as the fluorescent label and 2-methylpyridine borane complex (Sigma-Aldrich) as the catalyst. Each glycan received a single fluorescent label at the reducing terminus.…”

“…Fluorescently labeled glycans were separated by hydrophilic interaction liquid chromatography and measured by fluorescent detection (excitation wavelength: 330 nm, emission wavelength: 420 nm) according to the methods described by Valk-Weeber et al (2020b). The HPLC chromatograms obtained from acid whey protein glycoprofile analysis were integrated using Chromeleon software (version 6.8, Thermo Fischer Scientific, Waltham, MA), with each chromatogram divided manually into 32 individual peaks on a fixed baseline (Figure 1).…”

“…The glycan structures of all glycoproteins in the milk serum are represented in the obtained chromatograms. Previously, it was determined that the structures represented in the chromatogram originate primarily from GlyC-AM-1, lactoferrin, and IgG (Valk-Weeber et al, 2020b). Each of these major glycoproteins expresses a unique glycan fingerprint.…”

“…In dairy cattle, the glycosylation pattern of lactoferrin isolated from milk collected at 3, 30, and 90 DIM was demonstrated to be highly stable within a single cow as well as between cows (Valk-Weeber et al, 2020c). The glycoprofile of GlyCAM-1 appeared to be less stable, as variations in the sialylated and fucosylated glycans of the same cow were observed in milk collected at 30 and 90 DIM (Valk-Weeber et al, 2020b).…”