The Proteome Analysis Of Mfgm Original From Different Milk And The Apoptosis Mechanism Of Mfgm Proteins On Tumor Cells

Milk fat droplet is surrounded by milk fat globule membrane(MFGM),including proteins,phospholipids,amino acids,polysaccharides and various trace elements.MFGM also has a variety of biological functions.Presently,the research of MFGM proteome is on molecular level.Based on the understanding of the basic compositions and contents of five MFGM from bovine,goat,buffalo,yak and camel milk,consequently,the proteome of these five MFGM were studied by isotope labeling relative and absolute quantification(iTRAQ).The bioinformatics analysis was carried out on specific MFGM proteins.In addition,the inhibitory effects of five MFGM proteins on HT-29 colon cancer cells and MCF-7 breast cancer cells were studied.The compositions of MFGM from bovine,goat,buffalo,yak and camel milk were investigated in this study.The results revealed that the content of fat was highest in buffalo milk,whereas lowest in goat milk.There was a positive correlation between particle size of fat globule and fat contents.A significant difference(p<0.05)was observed in the compositions and contents of fatty acids from the five species milks.MFGM accounted for 2.07%~2.99% of the weight of fat globule.The protein contents of MFGM were highest in buffalo and yak milk.The polysaccharides of MFGM were highest in bovine milk,but lowest in yak milk.The mineral contents(P,Ca,K,Na)of the five MFGM were relati vely higher than other mineral contents.Moreover,the content of phosphorus was significantly higher in bovine and buffalo MFGM.The percentage of lipid contents of the five milk fat globule membrane proteins(MFGMPs)was between 53.64% to 66.34%.The polar lipid was higher in camel,bovine and buffalo MFGM than that from yak and goat MFGM.In addition,phospholipids were the major ingredient of polar lipid,which accounted for 85%~98%.iTRAQ technique was applied for the relative quantitative analysis of MFGMPs.The quantity of the proteins identified from bovine,goat,buffalo,yak and camel MFGM were 335,335,334,336 and 336,respectively.As the basis of bovine MFGMPs,goat and camel MFGMPs had the largest amount of up-regulating proteins,with quantities of 72 and 71,respectively.GO analysis results showed that the functions of MFGMPs were mainly catalytic activity and protein combination.They were mainly located in cell membrane and cytoplasm,involved in the metabolism process,stimulus response process,and biological process.Cluster results showed that the milks from the five species were organized into five major clusters: high-abundant proteins(Score > 1)of cluster I were 28 proteins,cluster II had 13 proteins,cluster III had 12 proteins,cluster IV had 9 proteins and cluster V had 18 proteins.Moreover,the cluster results of the KEGG of high-abundant proteins revealed that the 50% of pathways of these proteins were cancer related.PCA analysis(the total accumulative contribution rate of variance was over 85%)showed that the numbers of characteristic MFGMPs were 2 proteins from bovine MFGMPs,9 proteins from goat MFGMPs,5 proteins from buffalo MFGMPs,3 proteins from yak MFGMPs and 13 proteins from camel MFGMPs.Milk fat globule EGF factor VIII(MFG-E8),loner-chain acyl-Co Asynthctasc(ACSL),glycosylation-dependent cell-adhesion molecule1(Gly CAM1)and ATP-binding Cassette Sub-family G Member 2(ABCG2),which were from bovine,goat,buffalo and camel MFGMPs,were carried out through bioinformatics analysis.The results showed that there was no significant difference(p>0.05)between these four species.All four levels of amino acid sequence similarity were over 80%.Secondary structures of these four proteins all includes ?-helix,random coil and extending chain.All these four proteins belonged to irregular globular proteins.MFG-E8 protein has two N-glycosylation sites.Conserved domains of MFG-E8 were composed of two epidermal growth factors and two F5/8 type factor C dom ain structures.EGF is associated with cell proliferation and apoptosis,and F5/8C is for cell adhesion and lactation of mammary epithelial cells.ACSL3 includes two important functional domains which were ATP/AMP binding motifs and fatty acid binding motifs.The main functional structure domains of GlyCAM1,which has one N-glycosylation site and four O-glycosylation sites,were located in amino acid 20~152.Two functional structure domains of ABCG2 were ABCG-EPDR and ABC-2 type transpoter structural domain.ABCG-EPDR was associated with multidrug resistance and lipid transformation,and ABC-2 was responsible for transport protein activityThe inhibition of five MFGM on colon cancer cells(HT-29)and breast cancer cells(MCF-7)in vitro and their mechanisms were explored.MTT results showed that five MFGM inhibited the proliferation of both HT-29 and MCF-7 in a time and dose-dependent manner.Bovine,goat and buffalo MFGM blocked HT-29 in the S phases in a time-dependent manner.Goat,yak and camel MFGM blocked MCF-7 in the S phases.Bovine and buffalo MFGM blocked MCF-7 in the G2 phase,but the cell cycle did not change in a time-dependent manner.The results of morphology and flow cytometry showed that MFGM could induce the apoptosis of HT-29 and MCF-7.Rhodamine staining expressed that MFGM could reduce the activity of mitochondria in HT-29 and MCF-7 and induce apoptosis.Western blot showed that MFGM induced the apoptosis of HT-29 and MCF-7 through up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2.Among the five MFGM,goat and buffalo MFGM had greatest effect on HT-29,and buffalo MFGM had greatest effect on MCF-7.