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Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.

M Wuttke·S Müller·D P Nitsche·M Paulsson·F G Hanisch·P Maurer
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Abstract

Human bone sialoprotein (BSP) comprises 15% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodeling. Recent data suggest a role for BSP in breast cancer and the development of bone metastases. We have produced full-length recombinant BSP in a human cell line and purified the protein from human bone retaining the native structure with proper folding and post-translational modifications. Mass spectrometry of bone-derived BSP revealed an average mass of 49 kDa and for recombinant BSP 57 kDa. The post-translational modifications contribute 30-40%. Carbohydrate analysis revealed 10 different complex-type N-glycans on both proteins and eight different O-glycans on recombinant BSP, four of those were found on bone-derived BSP. We could identify eight threonines modified by O-glycans, leaving the C terminus of the protein free of glycans. The recombinant protein showed similar secondary structures as bone-derived BSP. BSP was visualized in electron microscopy as a globule linked to a thread-like structure. The affinity for hydroxyapatite was higher for bone-derived BSP than for recombinant BSP. Cell adhesion assays showed that the binding of BSP to cells can be reversibly diminished by denaturation.

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Amino Acid SequenceBlotting, WesternBone and BonesCarbohydrate SequenceCarbohydratesCell AdhesionCell LineChromatography, High Pressure LiquidChromatography, Ion ExchangeCircular DichroismDose-Response Relationship, DrugDurapatiteElectrophoresis, Polyacrylamide GelGlycosylationHumansIntegrin-Binding SialoproteinMass SpectrometryMethylationMicroscopy, ElectronMolecular Sequence DataOligosaccharidesPolysaccharidesProtein BindingProtein Processing, Post-TranslationalProtein Structure, SecondaryRecombinant ProteinsSialoglycoproteinsSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTime Factorsortho-AminobenzoatesJournal ArticleResearch Support, Non-U.S. Gov't

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M Wuttke, S Müller, D P Nitsche, M Paulsson, F G Hanisch, P Maurer. 2001-07-17. Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.. https://doi.org/10.1074/jbc.m105689200

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