Comparative N-glycoproteomics of Human and Bovine Whey and Milk Fat Globule Membrane During Lactation
Author:Cao Xue Zuo
Supervisor:yue xi qing
Breast milk contains all the nutrients needed of infants for their growth and development.The components of breast milk change during lactation to meet the requirements of infants at different growth times.Due to the importance of human milk for infants,the World Health Organization recommends exclusive breastfeeding during the first six months of life.Despite these advantages,there are still infants who cannot be breastfed.For them,infant formula is a good alternative to breast milk.To develop infant formula closer to breast milk,it is indispensable to study breast milk components thoroughly.Bovine milk is used widely as a dairy product and human milk substitute for infants.However,there are some differences between human and bovine milk.Therefore,to produce better dairy products and infant formulae,more similar to breast milk,it is necessary to eclucidate these differences between human and bovine milk.The short-term and long-term benefits of breastfeeding are probably due to the bioactive proteins in breast milk.Post-translational modifications(PTMs)influence the properties and functions of the modified proteins and increase their functions exponentially.Protein glycosylation is a common PTM that has pivotal roles in a variety of processes,including cell-cell recognition,signal transduction and immune defense.Elucidating glycoproteins and glycosites is the basis of study of protein functions.However,milk proteins glycosylation and its alteration during lactation are still little known.Casein is mostly phosphorylated.At present,the glycosylation of casein in human and bovine milk has been studied thoroughly.Thus,in the current study,we focused on the whey and milk fat globule membrane(MFGM)fractions.Using large-scale glycoproteomics,the quantitative differences of whey and MFGM N-glycoproteins between human colostrum and mature milk were investigated.Further,the comprehensive comparion of whey and MFGM N-glycoproteins between human and bovine milk during lactation were conducted.The results extend our knowledge of milk N-glycoproteome and the distribution of N-glycosylation sites in human and bovine milk,elucidate the potential functions of N-glycosylation on human and bovine milk protein from PTMs aspect,and provide insight into the biological functions of milk proteins and scientific basis for infant formula development.The main results are as follows:Using label-free quantitative glycoproteomics,133 N-glycosylation sites on 73 proteins were quantified in human milk whey.Among these,68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey.These differentially expressed N-glycoproteins were highly enriched in localization,extracellular region part,and modified amino acid binding according to gene ontology(GO)annotation analysis.In addition,16 immune-related differentially expressed glycoproteins were identified in human whey,most of which were upregulated in colostrum.(KEGG)pathway analysis revealed 33 pathways of the differentially expressed glycoproteins,and the major pathway was complement and coagulation cascades.Protein-protein interaction network analysis revealed some highly connected glycoproteins which might influence the entire system metabolism and signal transduction,including hypoxia upregulated protein 1 and haptoglobin.2.Using label-free quantitative glycoproteomics,912 N-glycosylation sites on 506 proteins were quantified in human milk MFGM.Among these,304 glycosylation sites on 220 proteins were differentially expressed in human colostrum and mature milk MFGM.These differentially expressed N-glycoproteins were involved in biological processes such as single-organism processes,biological regulation and regulation of biological processes;were cellular components in organelles and membranes;and had a mainly molecular function of protein binding.In addition,17 differentially expressed glycoproteins related to acute inflammatory response were identified in human MFGM,most of which were upregulated in colostrum.The KEGG pathway analysis revealed 155 pathways of the differentially expressed glycoproteins,mainly included phagosome,cell adhesion molecule and some disease-related pathways.Protein-protein interaction network analysis revealed some highly connected glycoproteins including pro-epidermal growth factor and fibronectin.3.Using comparative glycoproteomics,68,58,100,and 98 N-glycoproteins,and 111,96,167 and 139 N-glycopeptides were identified in human colostrum and mature milk as well as bovine colostrum and mature milk whey.GO annotation analysis revealed that the major whey glycoproteins were involved in single-organism processe in human colostrum and mature milk,were involoved in response to stimulus in bovine colostrum,and were involoved in biological regulation in bovine mature milk.Human and bovine colostrum and mature milk whey glycoproteins were mainly originated from extracellular region,cell,and organelle.The major molecular functions of human colostrum,human mature milk and bovine colostrum were protein binding,while were ion binding in bovine mature milk.The KEGG pathway analysis revealed 66,62,95 and 99 pathways of MFGM glycoproteins in human colostrum,human mature milk,bovine colostrum and bovine mature milk,respectively.Complement and coagulation cascades,Staphylococcus aureus infection and extracellular matrix-receptor interaction pathways were shared by human and bovine colostrum and mature milk.The comprehensive comparison of human and bovine whey glycoproteomes during lactation indicated more dramatic variations of whey N-glycosylation within species than lactation,only 9 N-glycoproteins were conserved in human and bovine colostrum and mature milk whey,while 9,4,34 and 36 N-glycoproteins were specific identified in human colostrum,human mature milk,bovine colostrum and bovine mature milk whey.4.Using comparative glycoproteomics,465,423,334,and 176 N-glycoproteins,and 843,718,614 and 273 N-glycopeptides were identified in human colostrum and mature milk as well as bovine colostrum and mature milk MFGM.GO annotation analysis revealed that the functional terms of four samples were similar.And the major MFGM glycoproteins were involved in cellular process,single-organism process,biological regulation,regulation of biological process,response to stimulus and metabolic process,other important processes including immune system process,location,signaling,and developmental process;were cellular components in cell,membrane and organelle;and had molecular functions of protein binding,receptor activitiy and hydrolase activity.The KEGG pathway analysis revealed 215,197,161 and 127 pathways of MFGM glycoproteins in human colostrum,human mature milk,bovine colostrum and bovine mature milk,respectively.Lysosome,phagosome,tuberculosis,complement and coagulation cascades,and cell adhesion molecules were shared by human and bovine colostrum and mature milk.Similar to whey N-glycoproteomes,the comprehensive comparison of human and bovine MFGM N-glycoproteomes during lactation indicated more dramatic variations of MFGM N-glycosylation within species than lactation,only 69 N-glycoproteins were conserved in human and bovine colostrum and mature milk,while 67,48,108 and 13 N-glycoproteins were specific identified in human colostrum,human mature milk,bovine colostrum and bovine mature milk MFGM.