Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties.
SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.
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