Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.
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.
Wuttke
M
M
Institute for Biochemistry II, Medical Faculty, University of Cologne, D-50931 Cologne, Germany.
Müller
S
S
Nitsche
D P
DP
Paulsson
M
M
Hanisch
F G
FG
Maurer
P
P
D000595:Amino Acid Sequence; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002448:Cell Adhesion; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D017886:Durapatite / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D013058:Mass Spectrometry; D008745:Methylation; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012795:Sialoglycoproteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D062367:ortho-Aminobenzoates / Q000378:metabolism
D002241:Carbohydrates; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D062367:ortho-Aminobenzoates; D017886:Durapatite; C000219:anthranilamide