The Interaction of Lactoferrin and Osteopontin and the Bioactivities of Lactoferrin-osteopontin Complex

Author:Liu Lan

Supervisor:liu jian xin bo lnnerdal


Degree Year:2019





Lactoferrin(Lactoferrin,LF)and osteopontin(Osteopontin,OPN)are both multi-functional whey proteins present at high levels in human milk.These two proteins have a high affinity to each other due to their opposite charges.LF and OPN were identified to bind to each other over a decade ago,but potential functions of their complex remain unexplored.Infant formula is based on bovine milk as the protein source and breast milk as the standard.To explore whether the complex composed by LF and OPN has the bioactivities thus to have the feasibility to be added into infant formula,the bioactivities of the human LF-OPN complex were first tested in vitro,including promotion of intestinal growth and development,inhibition of bacteria,and immune regulation.Then,the interaction of bovine LF and OPN were studied in details.The digestibility of bovine LF-OPN complex were explored both in vitro and in vivo by molecular biological methods.The effect of bovine complex on intestinal growth was especially studied.Finally,different proportions of bovine LF and OPN were added to the base powder of formula to test their bioactivities in vitro to explore the feasibility of adding LF and OPN into infant formula.1 Bioactivities of human LF-OPN complexThe native human LF-OPN complex was prepared by using LF and OPN extracted from breast milk.In vitro experiments were carried out to examine the characteristics of the LF-OPN complex with a focus on its bioactivities.LF and OPN coaccervated the most at pH 3.8,with a stronger stability of the LF-OPN complex during digestion than either protein.Moreover,the LF-OPN complex promoted proliferation of intestinal cells(HIEC)significantly more than the individual proteins(P<0.05).The complex promoted differentiation of Caco-2 cells significantly.Besides,the complex showed an intermediate effect on anti-bacterial function,which was reflective at inhibiton of the growth of E.coli and its adhesion of Caco-2 cells.The order of inhibiton was LF>the complex>OPN.For the immune regulation,the complex,LF and OPN significantly stimulated the secretion of IL-18 by Caco-2 cells(P<0.05),which also showed an intermediate effect.2 The interaction of bovine LF and OPN and the digestibility of the LF-OPN complexThe factors that influence the interaction between LF and OPN were examined.Firstly,the effects of pH,protein ratio,protein concentration,ionic strength,and temperature were detected.LF and OPN coaccervated the most at pH 3.8,consistent with the human complex result.And when the ratio of LF to OPN was 3:1,coaccervation happened the most.More colloids formed at higher concentration of the proteins and less ionic strength.Different temperature was examined at 4 ℃,37 ℃,and 60 ℃,respectively and the colloid formed at 37℃ was soluble.3 The effect of bovine LF-OPN complex on the proliferation of intestinal epithelial cells and the mechanism involvedThe bovine complexes showed a similar trend as human complex in digestion and promoting cell proliferation.Especially,the bovine complex(AH complex)composed by iron-free LF(apo-LF)and calcium binding OPN(holo-OPN)promoted cell proliferation the most,which has the similar form to the native LF and OPN in breast milk.Subsequently,the mechanism of the bovine complex on promoting cell proliferation was studied.Co-localization of the complex with the LF receptor and OPN receptor were found on the cell indicating that the complex may bind to the cell receptor and activate the corresponding signaling pathway.In addition,the complex entered the cell can still exist as a complex.It significantly promoted the activation of the PI3K/Akt signaling pathway compared to the two proteins alone.Inhibiton of the phosphorylation of Akt indicated that bovine complex promoted cell proliferation through PI3K/Akt signaling pathway.4 Preliminary study of bovine LF and OPN adding to infant formulaIn view of the biological activity of bovine LF-OPN complexes in promoting cell proliferation and surviving from digestion,three different ratios of LF and OPN were designed and the effects when making into infant formula were explored.The results of in vitro digestion showed that the ratio 3:1 of LF to OPN had the most significant effects on promoting of cell proliferation,anti-bacterial function and immune regulation(P<0.05).Thus,the ratio of LF and OPN is suggested to be added to infant formula.In summary,this study first proved that LF-OPN complex had the bioactivities of promoting intestinal growth and development,anti-bacterial functions,and immune regulation.Then,the characteristics of the interaction of LF and OPN were studied thus to optimize the conditions of preparing the complexes.Related bioactive function of bovine complex was focused on promting intestinal growth and its mechanism were revealed.This study provides essential evidence of the bioactivities of the LF-OPN complex and suggestions for the practical application of LF and OPN to improve infant formula.