PMC:2779105 / 84126-93878 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2779105","sourcedb":"PMC","sourceid":"2779105","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2779105","text":"Association of Fetuin-A and Albumin with the Mineral Phase of Nanoparticles\nThe seeding experiments shown here, comparing either untreated or boiled fetuin-A versus albumin in the presence of small submillimolar amounts of calcium and phosphate added to DMEM, demonstrate remarkable differences in dose-dependence, with fetuin-A showing much greater inhibitory activity than albumin. Along the same vein of observation, seeding appeared to be triggered at much lower doses of fetuin-A as compared to albumin. A functional inhibition assay developed in an earlier study also showed a 15-fold difference in inhibition between BSF and HSA (0.3 µM versus 6 µM, respectively) when the seeding experiment was carried out in aqueous solutions in the presence of 3 mM calcium-carbonate-phosphate ions (ref. [2]; see Fig. 19 of that study), a result that approximates the 14-fold difference seen with seeding experiments done in DMEM in the present study (Figs. 5– 7). In that same earlier study [2], both BSF and HSA were seen to bind to mineral complexes in a stoichiometric manner that could be demonstrated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; see Fig. 21 in ref. [2]). It is noteworthy that BSF and HSA that had been trapped by precipitating minerals gave similar intensities when analyzed by SDS-PAGE in spite of a 10-fold difference in their concentrations used, indicating again a much greater affinity of these mineral complexes for fetuin-A as compared to albumin (see Fig. 21 of ref. [2]). These results point to a hierarchy of protein binding by nascent apatite complexes that in turn can be predicted by the binding affinities of the individual proteins toward calcium and phosphate. Furthermore, they suggest that proteins present during particle formation may tentatively become an integral part of calcifying nanoparticles, a notion that had been advocated earlier to explain the biology of NB ([1]–[3], [46]; see also refs. [113], [114] for related studies).\nTo further ascertain that fetuin-A and albumin bind to calcium phosphate in a stoichiometric fashion and that these two proteins interact with each other by competing for apatite binding, we performed the following SDS-PAGE analysis. Specifically, we sought to use SDS-PAGE to demonstrate the marked differences in binding affinities between these proteins and apatite. Mineral complexes were formed in the presence of BSF, HSA, or both in DMEM inoculated with 3 mM calcium and phosphate as before; they were then washed and subjected to SDS-PAGE (Fig. 8A–C; see also Materials and Methods ). To facilitate referencing of the nanoparticles studied, we used in the Figure caption the acronym NLP, preceded by the name of the particular protein(s) used to form them (BSF, HSA, or both, described respectively in Figure 8A, B, and C). The NLP samples studied here were obtained by diluting fetuin-A and/or albumin into DMEM at the final concentrations of 20–160 µg/ml for BSF and/or 0.2–1.6 mg/ml for HSA, followed by the addition of the calcium and phosphate ions to a final concentration of 3 mM. Incubation was done in cell culture conditions for 1 month, during which time mineral precipitates appeared gradually that were then sedimented, washed, and processed for gel electrophoresis and Coomassie blue staining.\n10.1371/journal.pone.0008058.g008 Figure 8 Differences in the binding affinities between fetuin-A and albumin toward NB-like particles as revealed by SDS-PAGE.\nNB-like particles were prepared from 3 mM each of CaCl2 and NaH2PO4 in 1 ml of DMEM containing either BSF, HSA, or both proteins at the following concentrations: 20 µg/ml, 40 µg/ml, 80 µg/ml, or 160 µg/ml of BSF, corresponding respectively to lanes 1–4 in (A); 0.2 mg/ml, 0.4 mg/ml, 0.8 mg/ml, and 1.6 mg/ml of HSA, respectively, lanes 1–4 in (B); or both proteins at these same concentrations for lanes 1–4 in (C), respectively. Following incubation in cell culture conditions for 1 month, the NB-like particles were pelleted by centrifugation, washed twice in HEPES buffer, resuspended in 50 µl of 50 mM EDTA, and processed for SDS-PAGE as described in the Materials and Methods . In the case of BSF-NLP shown in (A), fetuin-A appeared as a major band slightly above 72 kDa (lane 1), with additional bands of higher and lower molecular weights noticeable at higher protein concentrations (lanes 2 to 4). In HSA-NLP (B), albumin formed a major band of strong intensity at 72 kDa. Note a higher molecular band above the 170 kDa marker that appears to increase steadily from lanes 1 through 4, while the 72 kDa band in the 4 lanes appears to decrease slightly in intensity from left to right. In the case of BSF-HSA-NLP (C), note the increase in the intensity of bands at 72 kDa and at a higher molecular weight. The bands of higher molecular weights may be due to altered migration or aggregation of the purified proteins used. In the case of BSF-NLP, an increase in protein band intensity was seen in direct proportion to the amount of BSF present in the incubation medium leading to mineral precipitation (Fig. 8A). Fetuin-A appeared initially as a single band slightly above 72 kDa (Fig. 8A, lane 1), but additional higher and lower bands could be distinguished when higher amounts of protein-mineral precipitate were electrophoresed under the same conditions (Fig. 8A, lanes 2–4). It should be pointed out that even though fetuin-A has a predicted molecular weight of 48 kDa, it is known to migrate to a higher molecular weight under denaturing and reducing conditions similar to what were used here, an observation that had been attributed earlier to the heavy glycosylation of this protein [115]. Furthermore, we have found that the migration of fetuin-A may vary with the commercial source of the lot as well as with the particular electrophoresis conditions used (for example, see Fig. 14 of ref. [2] for comparison). Notably, the higher molecular weight bands seen associated with fetuin-A (Fig. 8A, lane 4) were probably the result of aggregation in the presence of apatite, especially since control, untreated fetuin-A failed to show them (data not shown).\nIn the case of HSA-NLP, obtained using a 10-fold higher concentration of protein as compared to BSF-NLP (0.2–1.6 mg/ml for HSA vs. 20–160 µg/ml for BSF), all albumin-mineral samples analyzed showed a major band of 72 kDa. A dose-dependent increase in the intensity of this major band was not apparent, indicating that saturating doses of albumin must have been used here. On the other hand, with an increase in albumin input used for the formation of HSA-NLP, there was a noticeable dose-dependent increase of higher molecular weight bands (over 150 kDa) that appeared to correspond to aggregated/polymerized protein species (Fig. 8B, lanes 1–4). In fact, we chose to use these saturating amounts of albumin in order to assess more clearly the inhibition produced by fetuin-A, as follows.\nFigure 8C depicts the gel profile of various amounts of BSF-HSA-NLP formed in the presence of both BSF and HSA in the same amounts described above. There was a noticeable decrease in band intensity as compared with Figure 8B, indicating that albumin incorporation into the mineral complex had been significantly reduced. Since albumin had been used in excess in these experiments and exceeded the amount of fetuin-A by at least 10 fold, these results indicate clearly that the binding of fetuin-A to the mineral particles was not only significantly stronger than that seen for albumin, but that its binding to apatite appeared to compete off or inhibit the simultaneous binding by albumin. Nonetheless, it is also clear that both BSF and HSA appeared to have been incorporated into the particles, since the gel pattern seen with BSF-HSA-NLP appeared to incorporate characteristics of both BSF-NLP and HSA-NLP (compare the 3 gel profiles shown in Fig. 8). However, it is obvious from inspecting this gel profile that albumin only appeared to incorporate into the same mineral complexes at the higher doses used (0.8 and 1.6 mg/ml, lanes 3 and 4, Fig. 8C), whereas at the lower doses (0.2 and 0.4 mg/ml, lanes 1 and 2, Fig. 8C), only fetuin-A binding appeared to be more noticeable. Note also that in the presence of fetuin-A and albumin, the higher molecular weight bands appeared to migrate differently from those obtained with either protein alone—a result that perhaps suggests in further interactions between the two proteins when they were present together in the same complex. Given however that the two proteins migrated with overlapping molecular weights, the interpretation given here must be viewed with caution and must be considered along with the bulk of the functional data presented in the earlier sections in this study and elsewhere [2].\nThe data presented here show further that, on the one hand, proteins like fetuin-A and albumin appear to inhibit the formation of calcium nanoparticles, and, yet, they eventually incorporate into the same mineral nanoparticles that they attempt to inhibit in the first place. Moreover, our results clearly indicate that there is indeed a hierarchy in terms of preferential incorporation of proteins into the nanoparticle scaffold, a preference that is dictated by the differential binding affinities displayed by the proteins themselves. Fetuin-A, for one, binds more strongly to apatite minerals than albumin [2], [74], and thus it is a much more potent inhibitor of mineralization that in turn seems to interfere with concomitant albumin incorporation. Finally, these binding (inhibitory) processes appear to be transient, and, upon prolonged incubation, there is a gradual overcoming of this inhibition that yields to particle growth and seeding.","divisions":[{"label":"Title","span":{"begin":0,"end":75}},{"label":"Figure 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