Effects Of N-glycosylation On Physicochemical And Functional Properties Of Lactoferrin

Breast milk is the first choice for infant feeding.In recent years,the research and development of infant formula milk powder was committed to making milk powder close to breast milk in protein,fat,vitamins and other aspects,but clinical data showed that the nutritional function of breast milk was superior to that of milk powder.This causes people to pay attention to oligosaccharides in milk.Current studies mainly focus on free oligosaccharides in milk,but there are also some glycoprotein oligosaccharides in milk.Glycoprotein oligosaccharides are glycan molecules linked to protein molecules through glycosylation modification after protein translation,mainly N-glycosylation,endowing proteins with a wide range of biological functions,such as maintaining protein spatial structure,participating in receptor binding,regulating signal transduction between cells and immune regulation.However,current studies on the function of N-glycosylation had focused on the structural and biological functions of the in vivo bio-therapeutic glycoproteins.The effects of N-glycosylation on food-borne glycoproteins have not been extensively studied.Bovine lactoferrin is an immune glycoprotein in milk.It is a highly Nglycosylated protein with five N-glycosylated sites.As an exogenous protein,the effects of N-glycosylation on the structure and function of lactoferrin are still unknown.Therefore,this study systematically explored the effects of N-glycans on the physicochemical properties(structural stability,solubility and thermal stability)and functional properties(resistance to gastrointestinal digestion,regulation of protein absorption,absorption of intestinal epithelial cell,utilization of intestinal microorganisms,and bacteriostasis)of lactoferrin.In order to simulated the digestion,absorption and utilization of lactoferrin in the body,so as to reveal the importance of N-glycosylation to the biological functions of protein,and provide theoretical reference for the development of formula milk powder and functional foods in the future.The specific research contents and results are as follows:1.Structural identification of lactoferrin N-glycans and its effect on physicochemical properties of lactoferrinThe N-glycans structures of lactoferrin were identified.The changes of secondary and tertiary structure,protein solubility,thermal stability and antioxidant activity were measured before and after deglycosylation.A total of 17 N-glycans structures could be detected,including high-mannose,complex and hybrid-types.It can be seen that the highmannose structures are the major N-glycans of bovine lactoferrin,including Man-5 to Man-9.The content of high-mannose structures was about 65.7%.There are also complex-types including A2,A2G1,A2BG1,FA4,A2G2,FA3G1,FA2G2,A3G2S(6)and A2G2S(3)S(6).Hybrid-types included A1G1,M4A1G1 and M5A1G1.According to the peak area of each structure,the contents of sialylated structures and fucosylated structures in lactoferrin were about 4.4% and 1.1%,respectively(P>0.05).Deglycosylation did not affect the secondary structure of lactoferrin significantly.After deglycosylation,the fluorescence intensity,surface hydrophobicity and particle size of lactoferrin increased,and the protein was prone to aggregation.However,the solubility,thermal stability and antioxidant capacity of lactoferrin decreased significantly(P<0.05).Therefore,N-glycosylation played an important role in maintaining the structural stability of proteins,and the structure of deglycosylated proteins becomes unstable and prone to aggregation.2.Effect of N-glycosylation on lactoferrin digestion and its mechanismLactoferrin and deglycosylated lactoferrin were as the research materials to explore the effect of N-glycosylation on the digestive properties of lactoferrin.The results showed that the digestibility of lactoferrin and deglycosylated lactoferrin was only 3% in simulated infant gastric juice digestion,so most of lactoferrin entered into the intestine as an intact form.In simulated intestinal digestion,deglycosylated lactoferrin was more easily degraded by trypsin than lactoferrin.The protein digestibility of deglycosylated lactoferrin were significantly higher than that of lactoferrin(P<0.05).The ZDOCK dock results showed that the binding efficiency of trypsin and lactoferrin was improved after deglycosylation,and trypsin could more easily recognize the trypsin cutting site on lactoferrin.The difficulty of lactoferrin being degraded by trypsin might be related to the coverage of sugar chains on the structure.During the simulated digestion of adult gastric juice,lactoferrin and deglycosylated lactoferrin could be degraded by pepsin into smaller molecular fragments,and no complete protein entered into the intestine.Pepsin had no significant effect on the digestibility of lactoferrin and deglycosylated lactoferrin(P>0.05),which might be related to the wide specificity of pepsin digestion sites on lactoferrin.The in vitro digestibility of lactoferrin and deglycosylated lactoferrin was up to 94% in simulated gastrointestinal digestion.N-glycosylation had no significant effect on the in vitro digestibility of lactoferrin(P>0.05),but could affect the specific peptide composition of digestive product.Simulated gastrointestinal digestion did not change the structure of lactoferrin N-glycans,and N-glycans remained intact during gastrointestinal digestion.3.Effect of N-glycosylation on lactoferrin absorption and its mechanismThe Caco-2 cells monolayer model was used to study the effect of N-glycosylation on the absorption of lactoferrin and its digestive products.In addition,the absorption efficiency of digestion products of lactoferrin and free N-glycans by Caco-2 cells was measured.The results showed that deglycosylation could improve the ability of Caco-2 cells to absorb lactoferrin and its digestion products.The binding amount of deglycosylated lactoferrin to the membrane protein of Caco-2 cells was significantly higher than that of lactoferrin(P<0.05),but deglycosylation did not affect the expression of endocytosis related genes in Caco-2 cells(P>0.05).Docking results show that lactoferrin interacted with its receptor primarily through the peptide sequences Ser12-Arg38,Lys174-Tyr227 and Arg284-Leu298.However,the glycosylation modification sites Asn233 and Asn 281 are closely around Tyr227 and Arg284,Ser285,Phe286 and Gln287.Lactoferrin could expose to more amino acid sequences that interacted with its receptors,and the interaction between lactoferrin and the receptor was enhanced,leading to an increase in absorption after deglycosylation.Therefore,the effect of N-glycosylation on the absorption efficiency of lactoferrin by Caco-2 cells might be related to the improved binding efficiency of deglycosylated lactoferrin and receptor,but not to the expression level of endocytosis pathway related proteins.Caco-2 cells could not absorb free N-glycans and glycopeptides,or the absorption efficiency was much lower than that of intact proteins.4.Utilization mechanism of lactoferrin N-glycans by intestinal microorganismsLactoferrin and deglycosylated lactoferrin were used as substrates for the fermentation of intestinal microbiota in vitro.To elucidate the effects of N-glycosylation on fermentation characteristics of lactoferrin,and to speculate the utilization mechanism of N-glycans by intestinal microorganisms.The band of lactoferrin showed obvious displacement with the extension of fermentation time.The PAS results showed that N-glycans could be degraded and utilized by intestinal microorganisms during fermentation.N-glycans and various monosaccharides including galactose,mannose,N-acetylglucosamine,fucose and sialic acids could be gradually degraded and utilized by intestinal microorganisms.Acetic acid and propionic acid were the main products of lactoferrin N-glycans during fermentation in vitro.The N-glycans could significantly increase the contents of SCFAs in fermentation broth.Intestinal microorganisms could produce galactosidase,N-acetylglucosinase,fucosidase and mannosidase inside the cell and release them outside the cell.Both microbial fermentation broth and lysate could release part of N-glycans from lactoferrin,and could degrade the free N-glycans into smaller structures and monosaccharides,which could be metabolized and utilized by microorganisms or intestinal epithelial cells directly.Intestinal epithelial cells could produce galactosidase,N-acetylglucosidase,fucosidase,and mannosidase,which could not secrete outside of the cells.There were some differences in the utilization efficiency of monosaccharide between intestinal microorganisms and intestinal epithelial cells.The utilization efficiency of glucose and galactose by intestinal microorganisms was higher than N-acetylglucosamine and fucose,and the utilization efficiency of mannose was the lowest.Caco-2 cells absorbed glucose and mannose more efficiently than galactose,and almost did not absorb fucose and N-acetylglucosamine.5.Effect of N-glycosylation on lactoferrin antibacterial function and its mechanismListeria monocytogenes,Staphylococcus aureus,Escherichia coli O157 and Shigella were used as the target bacterias.The bacteriostasis and anti-biofilm activity of lactoferrin and deglycosylated lactoferrin were study.The iron binding capacity of lactoferrin and deglycosylated lactoferrin and their binding properties to four pathogenic bacteria were explore to speculate the role of N-glycans in antibacterial mechanism of lactoferrin.The effect of N-glycans on the adhesion effect of pathogenic bacteria to the Caco-2 cells also was determined.The results showed that lactoferrin had different inhibitory activities on the growth of four kinds of pathogenic bacteria,and had the best inhibitory effect on listeria monocytogenes.The bacteriostasis and anti-biofilm activity of lactoferrin were significantly weakened after deglycosylation,which might be related to the weakened binding ability of lactoferrin to iron ions or pathogenic bacteria.The presence of lactoferrin N-glycans could significantly weaken the adhesive ability of pathogenic bacteria to intestinal epithelial cells.In conclusion,N-glycans was essential for the antibacterial activity of lactoferrin.