α-乳白蛋白,大多数哺乳动物的乳汁中含有丰富的蛋白质,与生物有关,营养和技术功能。它的序列呈现N-糖基化基序,其占用是特定于物种的,从没有到完全占用。这里,我们调查了牛α-乳清蛋白在初乳和从四头奶牛取样的牛奶中的N-糖基化,每个在9个时间点从产牛当天开始到产后28.0d。使用以糖肽为中心的基于质谱的糖蛋白质组学方法,我们鉴定了在经典Asn-Xxx-Ser/Thr基序中发现的两个Asn残基上的N-糖基化,即分泌蛋白的Asn45和Asn74。我们在所有四头母牛中发现了相似的聚糖谱,部分现场占用,Asn45和Asn74的平均值分别为35%和4%。在这两个地点,哺乳期的入住率均未发生实质性变化。岩藻糖基化,唾液酸化,主要与N-乙酰神经氨酸(Neu5Ac),和高比例的N,N'-二乙酰基乳糖胺(LacdiNAc)/N-乙酰基乳糖胺(LacNAc)基序是已鉴定的N-聚糖的特征。虽然哺乳期间任何一个站点的站点占用都没有发生实质性变化,糖蛋白形式(即蛋白质的糖基化形式)谱显示出动态变化;从初乳到成熟乳的α-乳白蛋白糖蛋白形式库的成熟以中性聚糖和每个聚糖的LacNAc基序数量的大幅增加为标志,以牺牲LacdiNAc基序为代价。虽然α-乳清蛋白N-糖基化对功能性的影响尚不清楚,我们推测Asn74的N-糖基化导致结构和功能不同的蛋白质,由于与形成两个分子内二硫键的竞争。
α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based
glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N\'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.