The Regulation Of PepT2 On The Uptake Of Met-Met In Bovine Mammary Epithelial Cells

The objective of this study was to investigate the the mechanism of small peptide uptake and utilization in bovine mammary epithelial cells(BMECs).In Part 1,the mechanism of Met-Met promoted β-CN synthesis was examined.Part 2 was conducted to investigate the function of PepT2 in uptake of small peptide(Met-Met and β-Ala-Lys-AMCA)in BMECs.In Part 3,the effect of N-glycosylation and mammalian target of rapamycin(mTOR)on bovine peptide transporter 2(bPepT2)was explored.This study contributes to clarifying the mechanism of peptide utilization and improving milk quality in dairy cows.1.Met-Met promoted milk protein synthesis in BMECsThe objective of this study was to investigate utilization of Met-Met in milk protein synthesis in BMECs.BMECs were treated with different concentrations of Met-Met(0,20,40,80,120 and 160 μg/mL).In several experiments,the cells were treated with Janus kinase 2(JAK2)inhibitor(tyrphostin AG-490,50 μM)and mammalian target of rapamycin(mTOR)inhibitor(rapamycin,100 ng/mL).Met-Met dose-dependently increased the β-casein(P-CN)synthesis with an optimal concentration of 80 μg/ml in BMECs.At 80 μg/mL,Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from GO phase to S phase.In addition,80 μg/mL Met-Met increased the mRNA abundance of JAK2 and STAT5 and enhanced the phosphorylation of JAK2,signal transducer and activator of transcription 5(STAT5),mTOR,p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1.The inhibition of JAK2 and mTOR significantly decreased cell viability andβ-CN synthesis in BMECs.In summary,Met-Met enhanced milk protein synthesis by enhancing cell proliferation and activating JAK2-STAT5 and mTOR signaling pathways in BMECs.2.The expression and function of peptide transporter in bovine mammary glandThis study was conducted to investigate the expression and transport kinetics ofβ-alanyl-L-lysyl-Ns-7-amino-4-methyl-coumarin-3-acetic acid(β-Ala-Lys-AMCA)and Met-Met in BMECs.Polymerase chain reaction(PCR),western blotting and immunofluorescence were used to investigate the expression and location of the peptide transporters(PepTl,PepT2,PhTl,PhT2)in bovine mammary gland tissue.Uptake and transport studies were performed with P-Ala-Lys-AMCA and Met-Met-FITC.The effects of time,pH,concentration and inhibitors on p-Ala-Lys-AMCA and Met-Met uptake were studied.The results showed PepT2 and PhTl are expressed in bovine mammary gland by using PCR and western blotting.The immunofluorescence results showed that PepT2 and PhTl are both mainly located on the apical membrane and basolateral membrane of bovine mammary gland tissue.The uptake of P-Ala-Lys-AMCA was rapid during the first 60 minutes and reached plateau at 60 min.The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes.The optimal pH for the uptake ofβ-Ala-Lys-AMCA and Met-Met in BMECs was 6.5.The transport kinetic study suggested that the uptake of P-Ala-Lys-AMCA and Met-Met-FITC is saturable over the tested concentration.The transport of P-Ala-Lys-AMCA in BMECs exhibited a Michaelis constant(Km)of 82 μM and maximum transport velocity(Vmx)of 124 pmol/min/mg protein and for Met-Met-FITC Km is 52.4 μM and Vmax is 14.8 pmol/min/mg protein.The competitive inhibition results showed Gly-Sar,Met-Gly and Met-Met significantly inhibited P-Ala-Lys-AMCA uptake.However,histidine had no effect on P-Ala-Lys-AMCA uptake indicating that PhTl may not be involved in P-Ala-Lys-AMCA uptake.The uptake of Met-Met-FITC in BMECs was significantly inhibited by Met-Lys,Lys-Lys,Gly-Met,Gly-Leu and Met-Leu.Furthermore,knockdown of PepT2 significantly inhibited P-Ala-Lys-AMCA and Met-Met-FITC uptake,but PhTl inhibition has no effect on P-Ala-Lys-AMCA uptake.The above results showed that PepT2 may be involved in the uptake of small peptide in BMECs.3.The effects of N-Glycosylation in maintaining the transporter activity and expression of bPepT2The objective of this study was to investigate the effect of N-glycosylation on the expression and function of bPepT2.The bPepT2 gene was amplified,cloned and its nucleotide sequence was analysed.BioXM and DAS-TMfilter server software was used to predict amino acid composition and transmembrane domain of bPepT2,respectively.There are twelve transmembrane domains and a large extracellular loop between the ninth and tenth transmembrane domain in bPepT2.Five N-glycosylation sites exist in the large extracellular loop of bPepT2.The five N-glycosylation sites were mutated and cloned into pcDNA3.1 vector.Compared with the control group,the uptake of P-Ala-Lys-AMCA was significantly reduced after mutating the five N-glycosylation sites of bPepT2.Tunicamycin decreased the uptake of P-Ala-Lys-AMCA in transfected bPepT2 chinese hamster ovary cell(CHO)cells.Kinetic studies indicated that michaelis constant was not affected and maximum transport speed ofβ-Ala-Lys-AMCA in N-glycosylation mutant groups was significantly reduced compared to those of wild type.Immunofluorescence studies showed that the plasma membrane expression of bPepT2 was impaired in the mutant group.In summary,N-glycosylation influence function of bPepT2 by regulating the expression and subcellular location.4.The effect of mTOR on the expression and function of PepT2.This study was carried out to investigate the role of mTOR in regulating expression and activity of PepT2.Gene silencing,protein expression and peptide uptake studies were used to examine the role of mTOR in regulating the function of PepT2 and its underlying mechanism in BMECs.The results showed that rapamycin inhibited PepT2 expression and the uptake ofβ-Ala-Lys-AMCA in BMECs.Silencing DEPTOR(endogenous inhibitor of mTORC1 and mTORC2)promoted the expression of PepT2 in the cell membrane and the uptake of P-Ala-Lys-AMCA in BMECs.Both mTORC1 and mTORC2 silencing reduced the expression of PepT2 in the cell membrane and inhibited the uptake of β-Ala-Lys-AMCA in BMECs.Nedd4-2 silencing increased the trafficking of PepT2 to the plasma membrane and promoted the uptake of β-Ala-Lys-AMCA.With immunofluorescence double standard and Co-IP,PepT2 was identified to be interacted with Nedd4-2.mTORC1 inhibition increase the interaction between PepT2 and Nedd4-2 and promoted PepT2 ubiquination.mTORC1 interference increased Nedd4-2 expression.mTORCl and Nedd4-2 interference failed to decrease the expression and activity of PepT2 in the cell membrane.However,mTORC2 inhibition has no effect on Nedd4-2 expression and Nedd4-2 silencing did not influence the effect of mTORC2 inhibition on decreasing the expression and transport activity of PepT2 in BMECs.In summary,the results showed that mTOR can regulate the activity of PepT2.Both mTORC1 and mTORC2 are regulators of PepT2 and the regulation of PepT2 by mTORC1 is mediated by the Nedd4-2 in BMECs.In conclusion,Met-Met enhanced milk protein synthesis by enhancing cell proliferation and activating JAK2-STAT5 and mTOR signaling pathways in BMECs.PepT2 play an important role in the uptake of small peptide(β-Ala-Lys-AMCA and Met-Met).N-glycosylation existing in the large extracellular loop is important for bPepT2 and Nedd4-2 is required for mTORC1 regulation of PepT2 to regulate the uptake of small peptide in BMECs.