Phosphorylation and glycosylation isoforms of bovine κ-casein variant E in homozygous Swedish Red cow milk detected by liquid chromatography-electrospray ionization mass spectrometry

Abstract

Variations in the phosphorylation and glycosylation patterns of the common κ-casein (CN) variants A and B have been explored, whereas studies on variant E heterogeneity are scarce. This study reports for the first time the detailed phosphorylation and glycosylation pattern of the κ-CN variant E in comparison with variants A and B. Individual cow milk samples representing κ-CN genotype EE (n = 12) were obtained from Swedish Red cows, and the natural posttranslational modifications of its κ-CN were identified and quantified by liquid chromatography-electrospray mass spectrometry. In total, 12 unique isoform masses of κ-CN variant E were identified. In comparison, AA and BB milk consisted of 14 and 17 unique isoform masses, respectively. The most abundant κ-CN E isoform detected in the EE milk was the monophosphorylated, unglycosylated [1P 0G, ~70%; where P indicates phosphorylation from single to triple phosphorylation (1-3P), and G indicates glycosylation from single to triple glycosylation (1-3G)] form, followed by diphosphorylated, unglycosylated (2P 0G, ~12%) form, resembling known patterns from variants A and B. However, a clear distinction was the presence of the rare triphosphorylated, nonglycosylated (3P 0G, ~0.05%) κ-CN isoform in the EE milk. All isoforms detected in variant E were phosphorylated, giving a phosphorylation degree of 100%. This is comparable with the phosphorylation degree of variants A and B, being also almost 100%, though with very small amounts of nonphosphorylated, glycosylated isoforms detected. The glycosylation degree of variant E was found to be around 17%, a bit higher than observed for variant B (around 14%), and higher than variant A (around 7%). Among glycosylation, the glycan e was the most common type identified for all 3 variants, followed by c/d (straight and branched chain trisaccharides, respectively), and b. In contrast to κ-CN variants A and B, no glycan of type a was found in variant E. Taken together, this study shows that the posttranslational modification pattern of variant E resembles that of known variants to a large extent, but with subtle differences.

Effects of genetic variants and sialylation on in vitro digestibility of purified κ-casein

“…The distribution of isoforms in the purified materials was further assessed by LC-ESI/MS using ion intensities of the different isoforms to calculate relative abundances. The main isoforms, 1P0G, 2P0G, 1P1G (e), and 1P2G (e, e), were found to comply well with the isoform patterns recently reported for κ-CN A (n = 5), B (n = 4), and E (n = 12) molecules as present in individual skim milk (Thesbjerg et al, 2021;Sheng et al, 2022). This indicates that even though only a fraction of the collective pool of κ-CN molecules present in the used skim milk was purified from each milk source (AA, BB, and EE milk pools), the main isoform distribution of the purified fractions mirrors that of the native skim milk.…”

“…This indicates that even though only a fraction of the collective pool of κ-CN molecules present in the used skim milk was purified from each milk source (AA, BB, and EE milk pools), the main isoform distribution of the purified fractions mirrors that of the native skim milk. One minor difference was that, as shown in our previous work (Sheng et al, 2022), in milk, native κ-CN contained glycan types c, d, and e, as well as low abundances of a and b. However, only κ-CN with e, c, and d were identified in this study after purification, potentially due to their concentration being below the limit of detection.…”

“…What may be of greater significance relative to earlier reports regarding GD values for different protein forms of κ-CN may relate to its integration at UV level when analyzing complex samples such as skim milk. In earlier studies GD values of, for instance, 23 and 28% for variants A and B, respectively, as based on UV at 214 nm, have been reported (Poulsen et al, 2016), compared with 7, 14, and 17% as based on ion intensities (Sheng et al, 2022). This may indicate that the peak ascertained for glycosylated κ-CN might contain not only glycosylated κ-CN, but also some other proteins or protein fragments, and thus the quantification of κ-CN glycosylation calculated by LC and based on UV may be overestimated, whereas, using MS-based quantification for GD calculations, masses not complying with κ-CN are filtered off.…”

“…Different genetic variants may represent variances in posttranslational modifications. The glycosylation degree (GD), the ratio of glycosylated κ-CN to total κ-CN, and the glycan combinations all show high degrees of heterogeneity between genetic variants A, B, and E (Nilsson et al, 2020;Sheng et al, 2022). Overall, κ-CN variant B is known to have a higher GD than variant A, and recent reports indicate that variant E has a GD around or slightly higher than that of B (Jensen et al, 2015;Poulsen et al, 2016;Sheng et al, 2022).…”

“…The glycosylation degree (GD), the ratio of glycosylated κ-CN to total κ-CN, and the glycan combinations all show high degrees of heterogeneity between genetic variants A, B, and E (Nilsson et al, 2020;Sheng et al, 2022). Overall, κ-CN variant B is known to have a higher GD than variant A, and recent reports indicate that variant E has a GD around or slightly higher than that of B (Jensen et al, 2015;Poulsen et al, 2016;Sheng et al, 2022). Apart from glycosylation, bovine κ-CN is also known to be modified through disulfide bonds, with its 2 cysteine residues per κ-CN molecule (Cys 11 and Cys 88 ) being involved in intra-and intermolecular disulfide bonds (Mercier, Brignon, and Ribadeau-Dumas, 1973;Rasmussen et al, 1992).…”