Label Free Quantification Proteomics Reveals Novel Calcium Binding Proteins In Matrix Vesicles Isolated From Mineralizing Saos-2Cells

Skeletogenesis and bone development occur by a series of physicochemical andbiochemical processes that together facilitate the mineralization of the extracellularmatrix （ECM） at least in part by promoting the deposition of HA crystals in thesheltered interior of MVs. The MVs are membrane-invested bodies with a diameterranging from30to400nm and perform specialised roles in initiating matrixmineralization. These roles include transporting amorphous calcium phosphate（ACP）, managing mineral nucleation, regulating the Pi/PPi ratio in the intra-andextra-cellular fluid, controlling calcium ion and Pi homeostasis, and interacting withthe surrounding ECM to direct HA localisation and growth. The MVs possess aprotein and lipid machinery that is essential to carry out these functions, and they arehighly enriched in certain mineralization-relevant proteins. Recently, proteomicstudies of MVs isolated from different mineralization-competent cells have beenreported, but whole-proteome pattern analysis and quantified changes in MVs duringmineralization process are lacking.Human steosarcoma Saos-2cells undergo the entire osteoblastic differentiationsequence from proliferation to mineralization and release mineralization-competentMVs. Therefore, Saos-2cell, served as a model of pro-mineralizing cells, wereselected to analyze the functions of MVs. MVs are traditionally isolated fromconditioned media by serial ultracentrifugation. Recently a rapid proprietary method of exosomes isolation called ExoQuick^TM, which was reported as effective as theultracentrifugation method, has been made commercially available. To findadditional mineralization-relevant proteins, MVs were isolated from non-stimulatedand stimulated Saos-2cell cultures by ExoQuick^TMapproach and their proteomeswere identified and quantitatived by using label-free quantitative proteometechnology. The isolated MVs were confirmed by electron microscopy, alkalinephosphatase activity （ALP）, biomarkers, and mineral formation analyses. Thelabel-free quantitative proteome results showed that19calcium binding proteins（CaBPs）, such as Grp94, calnexin, calreticulin, calmodulin, and S100A4/A10, wereup-regulated in MVs of Saos-2cells upon stimulation for mineralization, which werepartly confirmed by western blotting. In this report, we provide the first evidencethat the ExoQuick^TMapproach is effective in isolating high quality MVs from Saos-2cells. Moreover, our data suggest that novel calcium binding proteins may beassociated with the initiation of MVs-mediated mineralization.Chapter1：AA and β-GP stimulated Saos-2cell mineralizationObjective：To examined the effect of AA and β-GP on Saos-2cell mineralization.Methods： Human osteosarcoma Saos-2cells were cultured in McCoy’s5Asupplemented with15%（v/v） fetal bovine serum,100U/ml penicillin and100μg/mlstreptomycin. The mineralization was induced by treatment of confluent Saos-2cellsfor6days in growth medium supplemented with7.5mM β-glycerophosphate and50μg/mL ascorbic acid. Calcium nodule analysis was performed by using AlizarinRed-S staining.Results：The calcium nodules were highly enhanced by the concomitant addition of50mg/ml AA and7.5Mm β-GP in Saos-2cells cultures. The stimulated Saos-2cellsproduced about6times more calcium nodules than non-stimulated Saos-2cellswithin6days.Conclusions：AA and β-GP are two osteogenic factors accelerating osteoblastic differentiation and mineralization. Chapter2：Isolation and validation of matrix vesiclesObjective：whether ExoQuick^TMapproach is effective in isolating high quality MVs.Methods：MVs were harvested from non-stimulated and stimulated Saos-2cellcultures at6days by using ExoQuick^TM. The isolated MVs were confirmed byelectron microscopy, biochemical （ALP activity）, biomarkers, and functional（mineral formation） analyses.Results：As seen by TEM, freshly isolated MVs were recognized as sphericalmembrane-bounded vesicle structures with a diameter ranging from30to400nm,and MVs isolated from stimulated cells contained electron dense material. Inaddition, MVs isolated from stimulated Saos-2cells revealed a higher ALP activitythan from the non-stimulated cells, as expected. Moreover, after incubation inmineralizing buffer for12h, MVs isolated from stimulated cells were able tocontinue uptaking Ca2+and form HA mineral as proved by the FTIR. Proteinsincluding CD63, CD9and Hsp70, known to be reliable biomarkers of the MVs andexosomes, were presented in both kinds of MVs preparationsConclusions：We first confirmed that the ExoQuick^TMapproach, which was used forthe isolation of biofluid and cell culture-derived exosomes, would be effective inisolating quality MVs from Saos-2cells. The isolated MVs from stimulated cells canstill uptake Ca ion and form HA when incubated in mineralizing buffer, whichindicated that MVs had its own proteins to carry out mineralization function. Chapter3：label-free quantitative proteome analysis of MVsObjective： To resolve subtle differences between proteomes of MVs fromnon-stimulated and stimulated Saos-2cells Methods;MVs were harvested from non-stimulated and stimulated Saos-2cell.Nano-flow LC-MS/MS experiment was performed on Easy-nLC1000equipped witha C18RP column. Analysis of tryptic peptides was performed using a Q-Exactivemass spectrometer. Continuum LC-MS data were processed and searched usingMaxQuant software （version1.3.0.5）. Quantitative analyses were performed using alabel-free approach. The significance of regulation level was determined at2foldchange.Results;We obtained255different proteins between MVs from non-stimulated andstimulated Saos-2cells by software analysis （fold≥2）;including186proteinsincreased whereas another69decreased.Conclusions;Label-free LC-MS/MS helped us to resolve subtle differences betweenMVs from non-stimulated and stimulated Saos-2cells. Chapter4：bioinformatical analysis of protein profiles in MVsObective：A bioinformatical analysis was performed to ananlyze the proteomicsdata of MVs from non-stimulated and stimulated Saos-2cells.Methods： Data analysis was completed by MAS3.0and http://string.embl.de/workstation. Five up-regulated proteins of label-free quantitative proteome resultswere selected for validation by western blotting.Results：Nineteen calcium binding proteins,6calmodulin binding proteins, and7proteins associated with calcium signaling pathway were up-regulated in MVs ofSaos-2cells upon stimulation for mineralization. STING workstation revealed thatthe calnexin, calreticulin and calmodulin act as the connection center among thecalcium-associated proteins. Consistent with label free quantification, four CaBPs（Grp94, calnexin, calregulin and S100A10） and ALP were detected and expressedhigher in the MVs isolated from stimulated cells than from non-stimulated cells.Conclusions：Together the proteomics and western blotting analysis, our data suggest that the results presented here represent reliable identification of the Saos-2cells’ MV proteomes. We highlight that new candidates among the CaBPs （Grp94,calnexin, calreticulin, calmodulin, and plastin-3） might play an important role inMVs-mediated initiation of mineralization.