Abstract
The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.
Highlights
The binding of native biglycan and decorin to pepsinextracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcoreTM2000 system
It was considered important that preparations of extracellular matrix macromolecules, e.g. decorin, classically obtained via a number of denaturing steps, sometimes show a weaker binding than recombinant, native protein expressed in eukaryotic cells, as shown in the case of decorin binding to collagen I (26)
Because denatured proteins may have an altered binding to their ligands, we used the native forms of biglycan and decorin, purified under mild conditions, for our binding studies
Summary
Mice deficient in biglycan show major alterations in bone (33). Biglycan can be extracted from, e.g. cartilage and purified under denaturing conditions. Such preparations have been used in studies of functional properties indicating that biglycan can inhibit binding of decorin to collagen VI (16). Native biglycan and decorin were shown to interact tightly via their core protein with the same binding site close to the N-terminal region of the collagen VI helical domain, possibly via the ␣2(VI). Native decorin was shown to bind to collagen VI with higher (10ϫ) affinity than has been shown previously for the molecule isolated under denaturing conditions (16)
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