Alteration of the N‐glycome of bovine milk glycoproteins during early lactation

Abstract

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc) ⁄ N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG 2 better than IgG 1 , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG 2 from the udder to blood, rather than to secrete IgG 1 into colostrum.

Bovine Milk Whey for Preparation of Natural N-glycans: Structural and Quantitative Analysis

“…A total of 39 N-glycans quantitatively detected from protein of bovine whey protein concentrate are composed of 26 neutral and 13 acidic sugars. N-Glycome was detected in bovine whole milk during the early lactation stage in a previous study [6]. In additional to general factors including diet, environment, seasons and species, N-glycan variations in bovine milk might have resulted from stages of lactation; a strong association between glycosylation and lactation period has been shown [6].…”

“…In additional to general factors including diet, environment, seasons and species, N-glycan variations in bovine milk might have resulted from stages of lactation; a strong association between glycosylation and lactation period has been shown [6]. Unlike glycoblotting and MALDI-TOF MS analysis [6], our 2D HPLC-mapping technique and MALDI-TOF MS analysis before and after exo-glycosidase sequential digestion enabled not only elucidation of the N-glycan composition but also determination of the detailed N-glycan structures including glycosidic linkage and isomeric monosaccharide discrimination. N-Glycans in bovine whey carried bi-or tri-antennae.…”

“…The Neu5Ac:Neu5Gc ratio varies among animal species, tissues and developmental stages. The molar ratio of Neu5Ac:Neu5Gc derived from bovine whey protein concentrate is 2.4; however, it has been suggested that bovine milk components may be altered depending on the lactation stage [6]. Differences in biological roles of Neu5Ac and Neu5Gc are less well understood.…”

“…The N-glycosylation profile of IgG, a major glycoprotein in bovine milk, was shown to be affected by  (2-3,6,8,9)-sialidase treatment but not by (2-3)-sialidase treatment, suggesting that the sialylated linkage on IgG is 2-6 [6]. This is in agreement with our finding that the α2-6-sialylated N-glycans derived from bovine milk protein concentrate are more abundant than α2-3-sialylated N-glycans by about 36.2 molar percent, and they are composed of Neu5Ac/ Neu5Gcα2-6Gal1-4GlcNAc and Neu5Acα2-6GalNAc1-4GlcNAc.…”

“…For example, alpha 1,2-linked fucosylated glycans found in human milk have been shown to inhibit binding of diarrhea-causing pathogens, including campylobacter, enterotoxigenic Escherichia coli and caliciviruses, and to protect breast-feeding infants from infectious diarrhea [5]. Analysis of N-glycans from bovine whole milk during the early lactation stage using a chemoselective glycoblotting technique and MALDI-TOF/TOF MS analysis revealed that the N-glycosylation profile of bovine milk glycoproteins changes over time in relation to N-glycosylation changes of IgG, which is a major immunoglobulin component present in bovine colostrum [6]. In this study, we analyzed the composition and structures of N-linked glycans derived from commercial whey protein concentrate by two-dimensional HPLC mapping in combination with MALDI-TOF/TOF MS techniques and quantitative comparison of N-glycans by a fluorescence-based technique that enables determination of low picomole quantities of N-glycans.…”