Milk osteopontin retains integrin-binding activity after in vitro gastrointestinal transit

Abstract

Osteopontin (OPN) is a multifunctional protein highly expressed in milk, where it is hypothesized to be involved in immunological signaling via the conserved Arg-Gly-Asp (RGD) integrin-binding sequence. Intervention studies have indicated beneficial effects of orally administered OPN in animal and human infants, but the mechanisms underlying these effects are not well described. To induce physiological effects, OPN must resist gastrointestinal transit in a bioactive form. In this study, we subjected bovine milk OPN to in vitro gastrointestinal transit, and characterized the generated fragments using monoclonal antibody and mass spectrometric analyses. We found that the fragment Trp 27 -Phe 151 containing the integrin-binding RGD sequence resisted in vitro gastric digestion. This resistance was dependent on glycosylation of threonine residues near the integrin-binding sequence in both human and bovine milk OPN. Furthermore, the fragment Trp 27 -Phe 151 retained the ability to interact with integrins in an RGD-dependent process. These results suggest a mechanism for how ingested milk OPN can induce physiological effects via integrin signaling in the intestine.

Factors influencing milk osteopontin concentration based on measurements from Danish Holstein cows

“…OPN has been shown to possess cytokine-like properties and regulate the Th1/Th2-type cytokine balanced immune response (Ashkar et al, 2000), induce the expression of interleukin-12 from intestinal mononuclear cells (Schack et al, 2009a,) and mark bacteria for opsonization by immune cells (Schack et al, 2009b). OPN is relatively resistant to proteolysis by gastric juices (Chatterton et al, 2004) and a bioactive fragment consisting of residues Trp 27 -Phe 151 has been shown to resist simulated gastrointestinal digestion in a form that enables interaction with integrins (Christensen et al, 2020), suggesting a mechanism for the beneficial effects of orally administered OPN reported in intervention studies.…”

Recombinant Bovine and Human Osteopontin Generated by Chlamydomonas reinhardtii Exhibit Bioactivities Similar to Bovine Milk Osteopontin When Assessed in Mouse Pups Fed Osteopontin‐Deficient Milk

“…[ 10,34 ] Previously, it has been shown that bmOPN survived proteolytic digestion in the stomach of 8–10 week old adult mice, [ 35 ] both bmOPN and hmOPN were resistant to in vitro digestion by human neonatal gastric juice, [ 36 ] and bmOPN and hmOPN subjected to in vitro digestion that simulated adult human gastrointestinal digestion yielded peptides that were able to bind to the integrin αVβ3 OPN receptor. [ 23 ] All OPNs in our study were relatively resistant to in vivo gastrointestinal digestion in P12 mouse pups, but as shown in Figure 4, a number of peptides were also formed. These peptides, as well as intact OPN, could possibly interact with OPN receptors on intestinal cells and thereby contribute to the multiple functions of OPN that promote intestinal development of infants.…”

“…Results from previous studies indicate that glycosylation plays a role in protecting OPN from digestion by pepsin and mediating phosphorylation of OPN. [ 23–25 ] The two algal recombinant OPNs used in this study are not glycosylated. Although rbOPN and rhOPN contain fewer phosphorylation sites and no glycosylation compared to bmOPN, there were no significant differences among the three supplementation groups (bmOPN, rbOPN, and rhOPN) in the functions tested for development of the brain, intestine, and immune system in mice, with the exception of a lower brain OPN level in the pups receiving recombinant OPNs.…”

“…For example, phosphorylation is required for effects of OPN on inhibition of vascular smooth muscle cell calcification, inhibition of calcium oxalate crystallization, and promotion of bone resorption. [ 20–22 ] Glycosylation of OPN has been documented to decrease pepsin digestion of OPN [ 23 ] and modify phosphorylation of OPN, [ 24,25 ] and thus also regulates availability and function of OPN.…”

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood–Brain Barriers

“…In vivo, the caseinomacropeptide (residues 106–169 of κ-casein) has been detected in plasma from newborns after breastfeeding and formula feeding, as well as in adult plasma after milk or yogurt ingestion [ 14 , 15 ]. The milk protein osteopontin resists gastric digestion [ 16 ], and osteopontin or fragments hereof have also been detected in the plasma of osteopontin-fed mice and infants [ 17 , 18 ].…”