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.

The glycoprotein osteopontin (OPN) possesses multiple functions in health and disease. To this end, osteopontin has beneficial roles in wound healing, bone homeostasis, and extracellular matrix (ECM) function. On the contrary, osteopontin can be deleterious for the human body during disease. Indeed, osteopontin is a cardinal mediator of tumor-associated inflammation and facilitates metastasis. The purpose of this review is to highlight the importance of osteopontin in malignant processes, focusing on lung and pleural tumors as examples.

To monitor the phosphorylation modifications and cellular localization of poly(rC)-binding protein-1 (PCBP1) during the cell cycle progression of Hela cells.

Hela cells highly synchronized at five different phases from interphase to mitosis were obtained. Using mitotic phosphoprotein-specific monoclonal antibody MPM-2, the exclusive occurrences of multiphosphorylation statuses of PCBP1 in mitosis were confirmed by a series of spots with increasing acidic pI (isoelectric point) in two rounds of 2D western blotting on the same membrane, and a visible molecular mass shift that can be eliminated by the treatment with λ phosphatase in 1D western blotting. Immnuofluorescence revealed the localization shift of PCBP1 during cell cycle, with accumulations in nucleus as a patch pattern in interphase, and a dispersive distribution without the area of the condensed chromosomes during mitosis.

These observations of mitosis-specific multiphosphorylations and localization shifts of PCBP1 suggest that the versatile PCBP1 was regulatable in a phosphorylation modification- and temporospatial-dependent manner in mitotic regulatory networks.

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 μg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

Osteopontin (OPN) is a multifunctional protein expressed in many cell types, tissues and body fluids with the highest concentrations found in milk; significantly higher in human than in bovine milk. Intervention studies have indicated beneficial effects of supplementing infant formula with bovine OPN. In this multicenter study, we determined the OPN content in human milk samples from 629 Chinese, Danish, Japanese and Korean mothers.

At each study site, milk samples were collected and analyzed for OPN and protein concentration using ELISA and infrared spectroscopy, respectively.

A total of 829 milk samples from 629 women were included. When delivering the first sample, mean maternal age was 31.4 years (SD 4.0), and median infant age was 13.4 weeks (interquartile range 4.6-17.9). The median OPN concentration varied across sites; from 99.7 mg/L in Danish, 185.0 mg/L in Japanese, 216.2 mg/L in Korean to 266.2 mg/L in Chinese mothers (P < 0.001), corresponding to 1.3%, 2.4%, 1.8% and 2.7% of the total protein content (OPN/protein%) (P < 0.05), respectively. Based on 75 Chinese and 33 Japanese mothers delivering more than 1 sample, multilevel (mixed model) linear regression analysis showed a decrease in OPN concentration with infant age (β = (-11.3), 95% confidence interval (CI) = (-13.9) to (-8.8) and β = (-2.1), 95% CI = (-3.2) to (-0.9), respectively).

In this large multicenter study, we observed statistically significant differences in the OPN concentration and the OPN/protein% in human milk samples between countries. Based on mothers delivering more than 1 sample, a significant decrease within the lactation period was observed.

Idiopathic calcium oxalate (CaOx) stone formers form stones that are commonly attached to calcium phosphate (CaP) deposits in the renal tissue, known as Randall's plaques (RP). Plaques are suggested to originate in the renal tubular basement membrane, where they exhibit a morphology of concentrically laminated apatitic spherules, while in the interstitial regions, the collagen fibrils and vesicles become mineralized. We hypothesize that these minerals might form by non-classical crystallization mechanisms, such as via amorphous precursors, some of which might originate from a polymer-induced liquid-precursor (PILP) process. Thus, our goal is to identify mineralogical 'signatures' of various stone formation mechanisms. To do this for idiopathic CaOx stones, we are developing a two-stage model system of CaP-CaOx composite stones, consisting of stage (1) CaP mineralized plaque, followed by stage (2) CaOx overgrowth into a stone. For the studies presented here, decellularized porcine kidneys were mineralized with CaP using polyaspartic acid or the protein osteopontin (OPN) to induce the PILP process and create biomimetic RP. Analysis of the PILP-mineralized tissues shows features that resemble the native plaques, including mineral spherules and collagen with intrafibrillar mineral. In contrast, the classical crystallization produced large apatitic spherulites, which is a very different morphology, but one which is also found in some stones. An alternative hypothesis regarding Randall's plaque, and if or when it becomes pathological, is discussed.

Drug-related kidney stones are a diagnostic problem, since they contain a large matrix (protein) fraction and are frequently incorrectly identified as matrix stones. A urine proteomics study patient produced a guaifenesin stone during her participation, allowing us to both correctly diagnose her disease and identify proteins critical to this drug stone-forming process. The patient provided three random midday urine samples for proteomics studies; one of which contained stone-like sediment with two distinct fractions. These solids were characterized with optical microscopy and Fourier transform infrared spectroscopy. Immunoblotting and quantitative mass spectrometry were used to quantitatively identify the proteins in urine and stone matrix. Infrared spectroscopy showed that the sediment was 60 % protein and 40 % guaifenesin and its metabolite guaiacol. Of the 156 distinct proteins identified in the proteomic studies, 49 were identified in the two stone-components with approximately 50 % of those proteins also found in this patient's urine. Many proteins observed in this drug-related stone have also been reported in proteomic matrix studies of uric acid and calcium containing stones. More importantly, nine proteins were highly enriched and highly abundant in the stone matrix and 8 were reciprocally depleted in urine, suggesting a critical role for these proteins in guaifenesin stone formation. Accurate stone analysis is critical to proper diagnosis and treatment of kidney stones. Many matrix proteins were common to all stone types, but likely not related to disease mechanism. This protocol defined a small set of proteins that were likely critical to guaifenesin stone formation based on their high enrichment and high abundance in stone matrix, and it should be applied to all stone types.

Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln(42) and Gln(193) serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln(42) predominantly is linked to Lys(4) and that Gln(193) participates in a cross-link with Lys(154), Lys(157), or Lys(231). The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys(154)-Gln(193) isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.

Objective. Previous studies have investigated the relationships between osteopontin gene polymorphism rs1126616 and OPN levels and urolithiasis, but the results were controversial. Our study aimed to clarify such relationships. Methods. A meta-analysis was performed by searching the databases Pubmed, Embase, and Web of Science for relevant studies. Crude odds ratios (ORs) or standardised mean differences with 95% confidence intervals (CIs) were calculated to evaluate the strength of association. Publication bias was estimated using Begg's funnel plots and Egger's regression test. Results. Overall, a significantly increased risk of urolithiasis was associated with OPN gene polymorphism rs1126616 for all the genetic models except recessive model. When stratified by ethnicity, the results were significant only in Turkish populations. For OPN level association, a low OPN level was detected in the urine of urolithiasis patients in large sample size subgroup. Results also indicated that urolithiasis patients have lower OPN level in serum than normal controls. Conclusion. This meta-analysis revealed that the T allele of OPN gene polymorphism increased susceptibility to urolithiasis. Moreover, significantly lower OPN levels were detected in urine and serum of urolithiasis patients than normal controls, thereby indicating that OPN has important functions in the progression of urolithiasis.

This article describes an updated computer model which attempts to simulate known renal reabsorption and secretion activity through the nephron (NEPHROSIM) and its possible relevance to the initiation of calcium-containing renal stones. The model shows that, under certain conditions of plasma composition, de novo nucleation of both calcium oxalate (CaOx) and calcium phosphate (CaP) can take place at the end of the descending limb of the Loop of Henle (DLH), particularly in untreated, recurrent idiopathic CaOx stone-formers (RSF). The model incorporates a number of hydrodynamic factors that may influence the subsequent growth of crystals nucleated at the end of the DLH as they progress down the renal tubules. These include the fact that (a) crystals of either CaOx or CaP nucleated at the end of the DLH and travelling close to the walls of the tubule travel at slower velocities than the fluid flowing at the central axis of the tubule, (b) the transit of CaOx crystals travelling close to the tubule walls may be delayed for up to at least 25 min, during which time the crystals may continue to grow if the relative supersaturation with respect to CaOx (RSS CaOx) is high enough and (c) such CaOx crystals may stop moving or even fall back in upward-draining collecting ducts (CD) owing to the Stokes gravitational effect. The model predicts, firstly, that for small, transient increases in plasma oxalate concentration, crystallisation only takes place in the CD and leads to the formation of small crystals which are comfortably passed in the urine and, secondly, that for slightly greater increases in the filtered load of oxalate, spontaneous and/or heterogeneous nucleation of CaOx may occur both at the end of the DLH and in the CD. This latter situation leads to the passage in the final urine of a mixture of large crystals of CaOx (arising from nucleation at the end of the DLH) and small crystals of CaOx (as a result of nucleation originating in the CD). As a result of the higher calcium and oxalate concentrations in the urine of RSF, these patients have an increased probability of initiating CaOx crystallisation in the DLH and so of going on to form the large crystals and aggregates found in their fresh urines, but not in the fresh urines from normal subjects (N). These predictions are supported by evidence from clinical studies on six RSF and six normal controls (NC) who were maintained for 4 days on a fixed basal diet. Their patterns of CaOx crystalluria were measured on the second day of the basal diet and after a small dose of sodium oxalate was given before breakfast on the fourth day of the study. The model also shows that the tubular fluid of RSF is more likely than that of N to reach the conditions necessary for de novo nucleation of CaP at the end of the DLH. This may occur following either a small increase in ultrafiltrable phosphate, as a result of ingestion of a high phosphate-containing meal, or a small decrease in the proximal tubular reabsorption of phosphate resulting, for example, from increased parathyroid activity. CaP crystals initiated at this point may heterogeneously nucleate the crystallisation of CaOx under the high metastable conditions of RSS CaOx which frequently exist in the urines of RSF. Under certain conditions, it is predicted that CaP crystals, initiated at the end of the DLH and travelling close to the tubular walls where their transit time is increased, might also be able to grow and agglomerate sufficiently to become trapped at some point in the CD and lead to the formation of Randall's Plugs in the Ducts of Bellini. Currently, work is under way to incorporate data on the growth and aggregation of crystals of CaP into NEPHROSIM to confirm the likelihood of this phenomenon occurring. The model shows that an increase in plasma calcium is unlikely to lead to spontaneous nucleation of either CaOx or CaP at the end of the DLH unless the concentration of plasma calcium reaches values usually associated with the cases of primary hyperparathyroidism. The most likely cause of spontaneous CaOx crystal formation at the end of the DLH is a small increase in plasma oxalate; the most likely cause of spontaneous CaP crystal formation at the end of the DLH is either an increase in plasma phosphate or a decrease in the fractional reabsorption of phosphate in the proximal tubule. The model predicts that the maximum volume of CaOx crystalluria that is likely to occur in a given urine is a function of both the RSS CaOx and the oxalate/calcium ratio in the final urine. These data explain why the volume of CaOx crystalluria is in the order UK normals < UK recurrent stone-formers < Saudi Arabian recurrent stone-formers which, in turn, probably accounts for the very high incidence of CaOx-containing stones found in Saudi Arabia compared with that in the UK.

Randall's plaque (RP) deposits seem to be consistent among the most common type of kidney stone formers, idiopathic calcium oxalate stone formers. This group forms calcium oxalate renal stones without any systemic symptoms, which contributes to the difficulty of understanding and treating this painful and recurring disease. Thus, the development of an in vitro model system to study idiopathic nephrolithiasis, beginning with RP pathogenesis, can help in identifying how plaques and subsequently stones form. One main theory of RP formation is that calcium phosphate deposits initially form in the basement membrane of the thin loops of Henle, which then fuse and spread into the interstitial tissue, and ultimately make their way across the urothelium, where upon exposure to the urine, the mineralized tissue serves as a nidus for overgrowth with calcium oxalate into a stone. Our group has found that many of the unusual morphologies found in RP and stones, such as concentrically laminated spherulites and mineralized collagenous tissue, can be reproduced in vitro using a polymer-induced liquid precursor (PILP) process, in which acidic polypeptides induce a liquid phase amorphous precursor to the mineral, yielding non-equilibrium crystal morphologies. Given that there are many acidic proteins and polysaccharides present in the renal tissue and urine, we have put forth the hypothesis that the PILP system may be involved in urolithiasis. Therefore, our goal is to develop an in vitro model system of these two stages of composite stone formation to study the role that various acidic macromolecules may play. In our initial experiments presented here, the development of "biomimetic" RP was investigated, which will then serve as a nidus for calcium oxalate overgrowth studies. To mimic the tissue environment, MatriStem(®) (ACell, Inc.), a decellularized porcine urinary bladder matrix was used, because it has both an intact epithelial basement membrane surface and a tunica propria layer, thus providing the two types of matrix constituents found associated with mineral in the early stages of RP formation. We found that when using the PILP process to mineralize this tissue matrix, the two sides led to dramatically different mineral textures, and they bore a striking resemblance to native RP, which was not seen in the tissue mineralized via the classical crystal nucleation and growth process. The interstitium side predominantly consisted of collagen-associated mineral, while the luminal side had much less mineral, which appeared to be tiny spherules embedded within the basement membrane. Although these studies are only preliminary, they support our hypothesis that kidney stones may involve non-classical crystallization pathways induced by the large variety of macromolecular species in the urinary environment. We believe that mineralization of native tissue scaffolds is useful for developing a model system of stone formation, with the ultimate goal of developing strategies to avoid RP and its detrimental consequences in stone formation, or developing therapeutic treatments to prevent or cure the disease. Supported by NIDDK grant RO1DK092311.