Effects Of Mechanical Strain On Glycoproteomics Analysis In Osteoblasts

The skeleton is a very complex tissue which can regulate its mass andarchitecture to adapt to functional environment. Orthodontic tooth movementresults from alveolar bone remodeling process induced by mechanical strain.The mechanical forces affect osteoblast on all respects，involving in cell shape，proliferation， structure and function. As the osteoblast is highly sensitive tomechanical stimulus，it plays the leading role in the bone remodeling process. Aproteomics approach，in which entire proteins in tissue or cells are identified andquantified directly，has been shown to be a valuable way to bring insight into themolecular basis of signal transduction. Compare the proteins of the osteoblastsin different conditions: mechanical strained and unstrained group，which willhelp us to find the key signal transduction protein and understand the responsivemechanism of the osteoblasts under the mechanical strain. Therefore in the studyof this part， we quantitatively and qualitatively analyzed the differentiallyexpressed proteins in MG-63osteoblastic cells after mechanical strain loading by lectin affinity chromatography and tandem mass spectrometry coupled toliquid chromatography (LC-MS/MS)， computer informatics and computernetwork communications technology.The aim of our study was to research progress of the effect of mechanicalstimulation on osteoblast. In the present study， MG-63osteoblastic cells weresubjected to mechanical strain using Flexcell strain loading system and observedthe glycoprotein glycan profiles of MG-63osteoblastic cells in differencesmechanical strain using lectin microarray. Furthermore，proteins from samplesin the MG-63osteoblastic cells obtained under mechanical strain which we’resubjected to lectin affinity chromatography and tandem mass spectrometrycoupled to liquid chromatography (LC-MS/MS) analysis. The research worksare as follows:1. Glycoprotein Glycan Analysis of MG-63osteoblastic-like cellsstimulated by mechanical strainAim: To analyze the glycoprotein glycan profiles of MG-63osteoblastic-like cells in differences mechanical strain.Methods: The total proteins of the MG-63osteoblastic cells of4h、8h、12hand24h after mechanical strain loading by using Flexcell strain loading systemand its control were extracted，labeled with Cy3fluorescent dye，then incubatedwith the lectin microarray. Glycoprotein Glycan profiles were analysed to screendifferently expressed Glycans，and got the relations of glycoprotein glycan andthe responsive mechanism of the osteoblasts under the mechanical strain.Results: The MG-63osteoblastic cells stimulated by mechanical strainshowed significant difference in lectin microarray compared with control samples.Conclusion: The lectin microarray could give the glycoprotein glycanprofiles and differences in glycan-profiles of the MG-63osteoblastic cells stimulated by differentially mechanical strain，and lectin microarray techniquewas effective to investigate mechanics of osteoblasts in bone remodeling.2. Glycoprotein purification using agarose bound lectinsAim: To prepare analysis of LC-MS/MS，we isolate glycoproteins of theMG-63osteoblastic cells under different conditions mechanical strain loadingby lectin affinity chromatography.Methods: Using agarose bound lectins of Jacalin and RCA120as solidsupporter enrich galactose-glyconjugates of the MG-63osteoblastic cells after8hand24h mechanical strain loading and its control. The immobilized lectin isallowed to bind to the glycoconjugate，and the unbound residual material can bereadily removed by subsequent washing during the purification ofglycoconjugates. The bound glycoconjugates are displaced from the immobilizedlectins by the addition of a solution of galactose to inhibit binding of the particularlectin. Through application of sodium dodecyl sulphate-polyacrylamide gelelectrophoresis verify enrichment effect.Results: Good separating effects were provided through application ofSDS-PAGE.Conclusion: Lectin immobilization method supported by agarose can beused as a tool to fraction the glycoproteins from complex samples and was usedin study of glycoprotein. It is simple and repid and may achieve the high andautomatization of identification of glycoproteins.3. LC-MS/MS analysis of differential expression protein in MG-63osteoblastic cells stimulated by mechanical strainAim: To establish an LC-MS/MS for determination of MG-63osteoblasticcells under mechanical strain loading and to clarify the major proteins involved in the molecular mechanism of osteoblasts under mechanical strain loading.Methods: Enrichment proteins were digest in solution. Ettan MDLCsystem was applied for desalting and separation of tryptic peptides mixtures. AFinnigan LTQ linear ion trap MS equipped with an electrospray interface wasconnected to the LC setup for eluted peptides detection. Data-dependent MS/MSspectra were obtained simultaneously. MS/MS spectra were automaticallysearched against the non-redundant International Protein Index (IPI) humanprotein database using the BioworksBrowser rev.3.1. Protein identificationresults were extracted from SEQUEST out files with BuildSummary.Results:24glycoproteins were obtained through LC-MS/MS analysis.These bone remodeling associated proteins fell into4groups, including energymetabolism(Alpha-enolase, Heat shock protein90-beta), cell proliferation(Galectin-1, Stress-70protein, reconstruction of cytoskeleton (CD44antigen,Tubulin alpha-1A chain) and signaling(Axin2，Voltage-dependent L-type calciumchannel subunit alpha-1D).Conclusion: Our study provided fundamental information on thedifferential expression of proteins of osteoblast under mechanical strain. Theseproteins described here may play important roles in mechanisms of biologicalbone remodeling process. These results are helpful for further study on themechanisms of bone remodeling and osteoblast formation under mechanicalstrain.