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

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 Trp27-Phe151 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 Trp27-Phe151 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.

Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVß3-integrin receptor.

Site-specific and partial proteolysis of milk proteins can both alter and increase their biological activity. The milk protein osteopontin (OPN) is a highly phosphorylated integrin-binding molecule present in most tissues and body fluids. Osteopontin is a biological substrate for matrix metalloproteinases, thrombin, plasmin, and cathepsin D. These proteases cleave OPN at several positions near the integrin-binding sequence Arg-Gly-Asp(138). This cleavage can either increase or reduce the ability of OPN to bind integrins and illustrates that small differences in the cleavage pattern can have a substantial effect on the functionality of OPN. Bovine milk OPN (bOPN) exists in both intact full-length and cleaved forms, and in this study, 6 N-terminal bOPN fragments originating from proteolytic cleavage were purified and characterized by mass spectrometry. These fragments were generated by cleavage at the Lys(145)-Ser(146), Arg(147)-Ser(148), Lys(149)-Lys(150), Phe(151)-Arg(152), Arg(152)-Arg(153), and Arg(153)-Ser(154) peptide bonds. The principal protease in milk, plasmin, appeared to cleave 3 of these sites. However, the major cleavage site was observed to be at the Phe(151)-Arg(152) bond, which does not match the specificity of plasmin. The bOPN fragments were shown to interact with the integrin receptor αVß3 as efficiently as the well-characterized and highly active OPN fragment Ile(1)-Arg(152), generated by thrombin cleavage of human milk OPN. These data show that OPN in milk is highly susceptible to proteolytic cleavage in the region containing the integrin-binding motifs, and the generated fragments are highly capable of binding cells via the αVß3-integrin.

Proteolytic activation of proteose peptone component 3 by release of a C-terminal peptide with antibacterial properties.

The milk protein proteose peptone component 3 (PP3, also known as lactophorin) is a small phosphoglycoprotein, which is exclusively expressed in the lactating mammary gland. A 23-residue synthetic peptide (lactophoricin, Lpcin S), corresponding to the C-terminal amphipathic α-helix of PP3, has previously been shown to permeabilize membranes and display antibacterial activity. Lactophorin readily undergoes proteolytic cleavage in milk and during dairy processing, and it has been suggested that PP3-derived peptides are part of milk's endogenous defense system against bacteria. Here, we report that a 26-residue C-terminal peptide (Lpcin P) can be generated by trypsin proteolysis of PP3 and that structural and functional studies of Lpcin P indicate that the peptide has antibacterial properties. The Lpcin P showed α-helical structure in both anionic and organic solvents, and the amount of α-helical structure was increased in the presence of lipid vesicles. Oriented circular dichroism showed that Lpcin P oriented parallel to the membrane surface. However, the peptide permeabilized calcein-containing vesicles efficiently. Lpcin P displayed antibacterial activity against Streptococcus thermophilus, but not against Staphylococcus aureus and Escherichia coli. The PP3 full-length protein did not display the same properties, which could indicate that PP3 functions as a precursor protein that upon proteolysis, releases a bioactive antibacterial peptide.

Considerable variation in the concentration of osteopontin in human milk, bovine milk, and infant formulas.

Osteopontin (OPN) is a multifunctional bioactive protein that is implicated in numerous biological processes such as bone remodeling, inhibition of ectopic calcification, and cellular adhesion and migration, as well as several immune functions. Osteopontin has cytokine-like properties and is a key factor in the initiation of T helper 1 immune responses. Osteopontin is present in most tissues and body fluids, with the highest concentrations being found in milk. In the present study, ELISA for human and bovine milk OPN were developed and OPN concentration in human breast milk, bovine milk, and infant formulas was measured and compared. The OPN concentration in human milk was measured to approximately 138 mg/L, which corresponds to 2.1% (wt/wt) of the total protein in human breast milk. This is considerably higher than the corresponding OPN concentrations in bovine milk (approximately 18 mg/L) and infant formulas (approximately 9 mg/L). Moreover, bovine milk OPN is shown to induce the expression of the T helper 1 cytokine IL-12 in cultured human lamina propria mononuclear cells isolated from intestinal biopsies. Finally, the OPN concentration in plasma samples from umbilical cords, 3-mo-old infants, and pregnant and nonpregnant adults was measured. The OPN level in plasma from 3-mo-old infants and umbilical cords was found to be 7 to 10 times higher than in adults. Thus, the high levels of OPN in milk and infant plasma suggest that OPN is important to infants and that ingested milk OPN is likely to induce cytokine production in neonate intestinal immune cells.

Osteopontin, a protein with cytokine-like properties, is associated with inflammation in Crohn's disease.

In Crohn's disease (CD) mucosal T-cells produce increased interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) levels and TNF-alpha antibody treatment [Infliximab (Ifx)] is effective. Osteopontin (OPN), a glycoprotein stimulating activated T-lymphocytes, may be involved in the disturbed immune-regulation but also in normal immune-homeostasis and mucosal repair, since it is expressed in many tissues and present in human milk. This study investigates plasma-OPN levels in CD patients during Ifx treatment and the in vitro effect of OPN on intestinal T cells. Thirty-seven CD patients received three Ifx doses at week 0, 2 and 6. Blood samples, colonic biopsies and clinical scores were obtained before treatment and at week 8, 26 and 52. In-vivo activated T-cell cultures were established from colonic biopsies in the presence of interleukin (IL)-2 and IL-4. The in vitro effect of OPN stimulation on T-cell IFN-gamma, TNF-alpha, and IL-10 production was measured. Plasma-OPN was increased in active CD (increased CRP-level) compared with quiescent disease (P = 0.02) and declined after three Ifx doses (P = 0.04). It was inversely correlated with in vitro T-cell IL-10 production. OPN increased CD69 and CD25 expression and enhanced T-cell IFN-gamma and TNF-alpha production in a dose-dependent fashion with higher levels in CD than in healthy controls (HC), but induced a concomitant higher IL-10 production in HC than CD. In conclusion, plasma-OPN levels are related to CD inflammation. In vitro, OPN-stimulated IL-10 production increases less in T-cell cultures from CD patients than from HC, indicating that IL-10 deficiency may be involved in the defect immune-regulation in CD, even after OPN stimulation.

Importance of phosphorylation for osteopontin regulation of biomineralization.

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a non-crystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca(2+). Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of beta-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.

SorCS3 does not require propeptide cleavage to bind nerve growth factor.

The functional properties of the Vps10p-domain receptor SorCS3 are undescribed. Here, we examine its processing and sorting in cellular transfectants, and analyze the binding of potential ligands to the purified receptor. We show that SorCS3 is synthesized as a proprotein and converted to its mature form by N-terminal propeptide cleavage in distal Golgi compartments. The propeptide is not a requirement for normal processing of the receptor and does not prevent ligands from binding to the SorCS3 precursor form. Expression of wt and chimeric receptors further suggests that SorCS3 predominates on the plasma membrane, exhibits slow internalization and does not engage in intracellular trafficking. SorCS3 emerges as a new neurotrophin binding Vps10p-domain receptor functionally distinct from its relatives Sortilin and SorLA.

Soluble osteopontin inhibits apoptosis of adherent endothelial cells deprived of growth factors.

Osteopontin (OPN) is primarily an extracellular glycosylated phosphoprotein capable of stimulating cell migration and cell attachment, predominantly to mineralized surfaces. Found in moderate levels in plasma, it acts as a cytokine able to modify gene expression via integrins and certain CD44 isoforms. In this work we show that soluble OPN inhibits apoptosis of adherent human umbilical vein endothelial cells incubated in medium lacking critical growth factors and cytokines. In a dose-dependent manner OPN reduced the formation of apoptotic bodies and suppressed DNA fragmentation. OPN also caused an increase in Bcl-X(L) mRNA levels, suppressed the apparent dispersion of Bcl-X(L) throughout the cytoplasm, and slightly enhanced IkappaB-alpha protein degradation. These data suggest that a function of OPN in homeostatic processes is to facilitate the survival of stressed endothelial cells, possibly by occupying unligated integrins and suppressing integrin-mediated death.

Recombinant expression of human mannan-binding lectin.

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Regulation of no synthesis induced by inflammatory mediators in RAW264.7 cells: collagen prevents inhibition by osteopontin.

Osteopontin has been shown to inhibit the induction of inducible nitric oxide synthase (iNOS, or NOS2) by lipopolysaccharide and interferon-gamma in the RAW264.7 mouse monocyte/macrophage line and in primary mouse proximal tubule epithelial cells. However, the RAW264.7 cells become refractory to the action of OPN after several subcultures or under dilute culture conditions, possibly because of changes in the composition of the extracellular matrix. We make this suggestion because if the cells are plated on a collagen type I or collagen type IV substrate the inhibitory action of OPN is completely suppressed; this is not the case on substrates consisting of laminin, fibronectin, poly-D-lysine, or poly-(2-hydroxyethylmethylacrylate). These observations imply that macrophages are sensitive to regulation by OPN only in certain physiological contexts. Both hyaluronate, which binds CD44, and rat IgGs are also able to inhibit the induction of NO synthesis by the inflammatory mediators. The similar actions of HA and OPN are consistent with the possibility that CD44 may be a receptor for OPN.

Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP.

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.

The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.

Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo.

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.

Localization of a single transglutaminase-reactive glutamine in the third domain of RAP, the alpha2-macroglobulin receptor-associated protein.

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln261 acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.

The primary structure of caprine PP3: amino acid sequence, phosphorylation, and glycosylation of component PP3 from the proteose-peptone fraction of caprine milk.

Proteose-peptone component 3 is a phosphorylated glycoprotein that was isolated from the proteose-peptone fraction of caprine milk. By mass spectrometric analysis, amino acid sequencing, and polymerase chain reaction analysis, the primary structure has been determined and has been shown to contain 136 amino acids. Phosphorylations were identified at Ser30 and Ser41. A partial glycosylation was present at Thr16, and a N-linked glycosylation was present at Asn78. Galactosamine was the amino sugar detected at Thr16. Glucosamine and galactosamine were the amino sugars found in the carbohydrate group linked to Asn78. The caprine amino acid sequence exhibits 88% identity with the bovine proteose-peptone component 3 sequence. However, when compared with the bovine sequence, the caprine sequence contains an insertion of a serine residue at position 25.

An examination of the binding of aluminum to protein and mineral components of bone and teeth.

Aluminum binding by a variety of noncollagenous proteins associated with bone and teeth (osteopontin, osteocalcin and phosphophoryn) and by hydroxyapatite are examined. The proteins bound aluminum with a dissociation constant, KD, of the 10(-7) M or greater at pH = 7. The number of atoms of aluminum bound was found to be related to but not equivalent to the number of phosphorylated serines in osteopontin and phosphophoryn. Osteocalcin bound one aluminum tightly presumably due to its gamma-carboxyl glutamate residues. Hydroxyapatite bound Al3+ tightly releasing 1.5 equivalent Ca2+ per Al3+ bound. Addition of 3 mM Ca2+, close to the total concentration found in animal circulating fluids, did not effect noticeably the amount of Al3+ bound to bone which must have a KD for Al3+ < 10(-6). Silicic acid added after equilibration with all these materials has little effect but neither the proteins nor hydroxyapatite removed much Al3+ from pre-equilibrated Al3+ solution with silicic acid. The results are discussed with regard to Al3+ poisoning.

Characterization of phosphate sites in native ovine, caprine, and bovine casein micelles and their caseinomacropeptides: a solid-state phosphorus-31 nuclear magnetic resonance and sequence and mass spectrometric study.

The phosphate sites in native ovine, caprine, and bovine casein micelles have been analyzed using sequence analysis, mass spectrometric analysis, and solid-state 31P nuclear magnetic resonance spectroscopy. Using a combination of S-ethylcysteine derivatization, sequence analysis, and mass spectrometric analysis, the phosphorylation sites of ovine (SerP151 and SerP168), caprine (SerP151 and SerP168), and bovine (SerP149) caseinomacropeptides have been localized. Various solid-state 31P methods using magic angle spinning have been applied to ascertain the local structure and dynamics of the phosphorylated serine residues and the inorganic calcium phosphates within the micelles. Contributions from the phosphorylated serine residues of kappa-CN, located in the C-terminal portion of the molecule, to the mobile constituents of the micelles were assigned by comparison with 31P nuclear magnetic resonance spectra of purified caseinomacropeptides from the various species in the dissolved state. Comparison of the 31P magic angle spinning nuclear magnetic resonance spectra of ovine, caprine, and bovine casein micelles indicates that the micelles from these species are very similar but not identical.

The localization and multimeric nature of component PP3 in bovine milk: purification and characterization of PP3 from caprine and ovine milks.

The distribution of proteose-peptone component PP3 in bovine whey, milk fat globule membrane, and casein has been investigated with antibodies raised against highly purified PP3. Using Western blot analysis, we show that PP3 is present in the milk fat globule membrane and in whey but is absent in the casein fraction. The proposed multimeric structure of bovine PP3 was analyzed by mass spectrometry and gel filtration. Calibrated gel filtration of acidic whey showed that PP3 eluted at a volume corresponding to 190 kDa, indicating that PP3 exists as a multimeric aggregate in bovine milk. Western blot analysis with anti-bovine PP3 immunoglobulins was used to analyze caprine, ovine, and human milks, and immunoreactive proteins were detected in caprine and ovine milks. Finally, the immunoreactive proteins from caprine and ovine milks were purified and characterized as PP3 analogues by amino acid analysis and N-terminal sequence analysis.