牛初乳和牛常乳乳清N-糖蛋白质的差异分析
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摘要
为阐明牛乳蛋白质N-糖基化,采用糖蛋白质组学技术,在牛初乳和牛常乳乳清中共鉴定到154个N-糖蛋白和246个糖基化位点,其中,牛初乳鉴定到117个N-糖蛋白和183个糖基化位点;牛常乳鉴定到109个N-糖蛋白和145个糖基化位点。初乳中丛生蛋白(P17697)糖基化位点N-283表达量最高,常乳中α-乳白蛋白糖基化位点N-93表达量最高。考虑定量差异及有无差异,在牛初乳和牛常乳乳清中共鉴定到129个糖蛋白的190个差异表达糖基化位点。基因本体论功能注释表明,差异表达糖蛋白参与的生物学过程是生物调节、刺激性反应、多细胞生物过程、定位、免疫系统过程等;主要分布为细胞外区域和细胞器;主要的分子功能是结合作用、催化活性和分子功能调节。差异表达糖蛋白参与的代谢通路主要是补体与凝血级联、金黄色葡萄球菌感染和溶酶体等。此外,通过蛋白互作分析,找到一些具有高连接度的重要糖蛋白。本研究丰富了牛乳N-糖蛋白质组成及其糖基化位点信息,阐明了牛乳乳清N-糖基化的功能,为评价和改善牛乳品质、研发婴幼儿配方乳中糖蛋白质的改良及功能食品的生产提供了理论依据。
Protein glycosylation is one of the most important post-translational modifications, which influences the functions of the modified proteins. The aim of this study was to elucidate the N-glycosylation of proteins in bovine milk. Using glycoproteomics, a total of 154 N-glycoproteins with 246 glycosylation sites were identified in bovine milk, including 117 N-glycoproteins with 183 glycosylation sites in colostrum, and 109 N-glycoproteins with 145 glycosylation sites in mature milk. The glycosylation site of N-283 on clusterin was expressed at the highest level in colostrum while N-93 on alphalactalbumin showed the highest expression in mature milk. Considering the quantitative and exclusive differences, 190 glycosylation sites on 129 glycoproteins were differentially expressed between colostrum and mature milk. According to gene ontology annotation, these differentially expressed glycoproteins participated in biological regulation, response to stimulus, multicellular organismal processes, localization and immune system processes; they were cellular components in the extracellular region and organelles and had various molecular functions such as binding, catalytic activity and molecular function regulators. The differentially expressed glycoproteins were mainly involved in the complement and coagulation cascades, Staphylococcus aureus infection and the lysosome pathways. In addition, some highly connected glycoproteins were identified through protein-protein interaction analysis. Our results enrich the knowledge on the glycoproteome of cow's milk, including the N-glycoprotein composition and glycosylation sites and also reveal the functions of protein N-glycosylation in bovine milk whey, which provide a theoretical foundation for the evaluation and improvement of bovine milk quality, the development of infant formula closer to human milk, and the production of functional foods.
Protein glycosylation is one of the most important post-translational modifications, which influences the functions of the modified proteins. The aim of this study was to elucidate the N-glycosylation of proteins in bovine milk. Using glycoproteomics, a total of 154 N-glycoproteins with 246 glycosylation sites were identified in bovine milk, including 117 N-glycoproteins with 183 glycosylation sites in colostrum, and 109 N-glycoproteins with 145 glycosylation sites in mature milk. The glycosylation site of N-283 on clusterin was expressed at the highest level in colostrum while N-93 on alphalactalbumin showed the highest expression in mature milk. Considering the quantitative and exclusive differences, 190 glycosylation sites on 129 glycoproteins were differentially expressed between colostrum and mature milk. According to gene ontology annotation, these differentially expressed glycoproteins participated in biological regulation, response to stimulus, multicellular organismal processes, localization and immune system processes; they were cellular components in the extracellular region and organelles and had various molecular functions such as binding, catalytic activity and molecular function regulators. The differentially expressed glycoproteins were mainly involved in the complement and coagulation cascades, Staphylococcus aureus infection and the lysosome pathways. In addition, some highly connected glycoproteins were identified through protein-protein interaction analysis. Our results enrich the knowledge on the glycoproteome of cow's milk, including the N-glycoprotein composition and glycosylation sites and also reveal the functions of protein N-glycosylation in bovine milk whey, which provide a theoretical foundation for the evaluation and improvement of bovine milk quality, the development of infant formula closer to human milk, and the production of functional foods.
引文
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[24]HUANG J C,GUERRERO A,PARKER E,et al.Site-specific glycosylation of secretory immunoglobulin A from human colostrum[J].Journal of Proteome Research,2015,14(3):1335-1349.DOI:10.1021/pr500826q.
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[29]赵菲,党刘毅,赵璇,等.补体系统及其糖基化[J].生物化学与生物物理进展,2017,44(10):888-897.DOI:10.16476/j.pibb.2017.0320.
[30]D’ALESSANDRO A,SCALONI A,ZOLLA L.Human milk proteins:an interactomics and updated functional overview[J].Journal of Proteome Research,2010,9(7):3339-3373.DOI:10.1021/pr100123f.
[2]包晓宇,陈美霞,王加启,等.牛乳中活性蛋白生物学功能研究进展[J].食品科学,2017,38(19):315-324.DOI:10.7506/spkx1002-6630-201719049.
[3]刘晶,韩清波.乳清蛋白的特性及应用[J].食品科学,2007,28(7):535-537.
[4]蒲玲玲,郭长江.乳清蛋白的组成及其主要保健功能[J].中国食物与营养,2011,17(6):68-70.
[5]YANG M,CAO X Y,WU R N,et al.Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS[J].International Journal of Food Sciences&Nutrition,2017,68(6):671-681.DOI:10.1080/09637486.2017.1279129.
[6]YANG M,CONG M,PENG X M,et al.Quantitative proteomic analysis of milk fat globule membrane(MFGM)proteins in human and bovine colostrum and mature milk samples through iTRAQlabeling[J].Food&Function,2016,7(5):2438-2450.DOI:10.1039/c6fo00083e.
[7]LU J,WANG X Y,ZHANG W Q,et al.Comparative proteomics of milk fat globule membrane in different species reveals variations in lactation and nutrition[J].Food Chemistry,2016,196:665-672.DOI:10.1016/j.foodchem.2015.10.005.
[8]YANG Y Y,BU D P,ZHAO X W,et al.Proteomic analysis of cow,yak,buffalo,goat and camel milk whey proteins:quantitative differential expression patterns[J].Journal of Proteome Research,2013,12(4):1660-1667.DOI:10.1002/pmic.201500361.
[9]YU L Y,HE H B,HU Z F,et al.Comprehensive quantification of N-glycoproteome in Fusarium graminearum reveals intensive glycosylation changes against fungicide[J].Journal of Proteomics,2016,142:82-90.DOI:10.1016/j.jprot.2016.05.011.
[10]ZIELINSKA D F,GNAD F,WI?NIEWSKI J R,et al.Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints[J].Cell,2010,141(5):897-907.DOI:10.1016/j.cell.2010.04.012.
[11]FROEHLICH J W,DODDS E D,BARBOZA M,et al.Glycoprotein expression in human milk during lactation[J].Journal of Agricultural&Food Chemistry,2010,58(10):6440-6448.DOI:10.1021/jf100112x.
[12]CAO X Y,KANG S M,YANG M,et al.Quantitative N-glycoproteomics of milk fat globule membrane in human colostrum and mature milk reveals changes in protein glycosylation during lactation[J].Food&Function 2018,9:1163-1172.DOI:10.1039/C7FO01796K.
[13]YANG Y X,ZHENG N,ZHAO X W,et al.N-glycosylation proteomic characterization and cross-species comparison of milk whey proteins from dairy animals[J].Proteomics,2017,17(9):1600434.DOI:10.1002/pmic.201600434.
[14]CAO X Y,SONG D H,YANG M,et al.Comparative analysis of whey N-glycoproteins in human colostrum and mature milk using quantitative glycoproteomics[J].Journal of Agricultural&Food Chemistry,2017,65:10360-10367.DOI:10.1021/acs.jafc.7b04381.
[15]YANG Y X,ZHENG N,WANG W Y,et al.N-glycosylation proteomic characterization and cross-species comparison of milk fat globule membrane proteins from mammals[J].Proteomics,2016,16(21):2792-2800.DOI:10.1002/pmic.201500361.
[16]曹雪妍,杨梅,岳喜庆.乳蛋白质糖基化的研究进展[J].乳业科学与技术,2018,41(1):40-46.DOI:10.15922/j.cnki.jdst.2018.01.008.
[17]NAGARAJA B,NISHIMURA S I,SOSALEGOWDA A H.A comparative glycomics of fat globule membrane glycoconjugates from Buffalo(Bubalus bubalis)milk&colostrum[J].Journal of Agricultural&Food Chemistry,2017,65(8):1496-1506.DOI:10.1021/acs.jafc.6b03330.
[18]YANG M,SONG D H,CAO X Y,et al.Comparative proteomic analysis of milk-derived exosomes in human and bovine colostrum and mature milk samples by iTRAQ-coupled LC-MS/MS[J].Food Research International,2017,92:17-25.DOI:10.1016/j.foodres.2016.11.041.
[19]GENG F,WANG J Q,LIU D Y,et al.Identification of N-glycosites in chicken egg white proteins using an omics strategy[J].Journal of Agricultural&Food Chemistry,2017,65(26):5357-5364.DOI:10.1021/acs.jafc.7b01706.
[20]FANG P,WANG X J,XUE Y,et al.In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins[J].Oncotarget,2016,7(25):38796-38809.DOI:10.18632/oncotarget.9737.
[21]杨梅.不同泌乳期人乳与牛乳中非酪蛋白差异蛋白质组学研究[D].沈阳:沈阳农业大学,2017:32-37.
[22]LE A,DOUGLAS BARTON L D,SANDERS J T,et al.Exploration of bovine milk proteome in colostral and mature whey using an ionexchange approach[J].Journal of Proteome Research,2011,10(2):692-704.DOI:10.1021/pr100884z.
[23]乌兰君,杨梅,王满霞,等.牛初乳与牛乳中乳清蛋白质组成及功能的对比分析[J].现代食品科技,2017,33(5):58-63.DOI:10.13982/j.mfst.1673-9078.2017.5.010.
[24]HUANG J C,GUERRERO A,PARKER E,et al.Site-specific glycosylation of secretory immunoglobulin A from human colostrum[J].Journal of Proteome Research,2015,14(3):1335-1349.DOI:10.1021/pr500826q.
[25]MCDONALD J F,NELSESTUEN G L.Potent inhibition of terminal complement assembly by clusterin:characterization of its impact on C9 polymerization[J].Biochemistry,1997,36(24):7464-7473.DOI:10.1021/bi962895r.
[26]朱凌燕,朱晗,王军波.α-乳白蛋白对婴儿生长发育影响的研究现况[J].中国生育健康杂志,2015(2):193-196.DOI:10.3969/j.issn.1671-878X.2015.02.030.
[27]余英才,张纯,夏循礼,等.补体系统的进化[J].生命科学,2012,24(4):362-367.DOI:10.13376/j.cbls/2012.04.014.
[28]MAYE S,STANTON C,FITZGERALD G F,et al.Detection and characterisation of complement protein activity in bovine milk by bactericidal sequestration assay[J].Journal of Dairy Research,2015,82(3):328-333.DOI:10.1017/S0022029915000266.
[29]赵菲,党刘毅,赵璇,等.补体系统及其糖基化[J].生物化学与生物物理进展,2017,44(10):888-897.DOI:10.16476/j.pibb.2017.0320.
[30]D’ALESSANDRO A,SCALONI A,ZOLLA L.Human milk proteins:an interactomics and updated functional overview[J].Journal of Proteome Research,2010,9(7):3339-3373.DOI:10.1021/pr100123f.