Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding
| Autor: | M, Wuttke, S, Müller, D P, Nitsche, M, Paulsson, F G, Hanisch, P, Maurer |
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| Rok vydání: | 2001 |
| Předmět: |
Glycosylation
Time Factors Sialoglycoproteins Blotting Western Molecular Sequence Data Carbohydrates Oligosaccharides Methylation Bone and Bones Mass Spectrometry Protein Structure Secondary Cell Line Polysaccharides Cell Adhesion Humans Integrin-Binding Sialoprotein ortho-Aminobenzoates Amino Acid Sequence Chromatography High Pressure Liquid Dose-Response Relationship Drug Circular Dichroism Chromatography Ion Exchange Recombinant Proteins Microscopy Electron Durapatite Carbohydrate Sequence Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Electrophoresis Polyacrylamide Gel Protein Processing Post-Translational Protein Binding |
| Zdroj: | The Journal of biological chemistry. 276(39) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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