Zur molekularen Topologie der Bindung natürlicher und rekombinanter Varianten von Alpha 2 -HS-Glycoprotein-Feturin an Hydroxylapatit
The plasma protein alpha-2-HS-glycoprotein (AHSG)/fetuin transiently inhibits the calcium phosphate precipitation from supersaturated solution. The unique structural motif within the first domain of AHSG mediating the inhibition of precipitation was studied by use of structure-function relations of protein domains of the cystatin superfamily. Since no structural data of these cystatin-like domains were available, models were generated by comparative structure modelling. The model of AHSG domain D1 shows an aminoterminal alpha-helix and a bent four-stranded beta-sheet. Seven acidic amino acids form a contiguous and negatively charged surface area. No other cystatin-like domain contains a comparable negatively charged surface area, suggesting that the inhibition is mediated by an interaction of these acidic residues with calcium ions on the mineral surface. The effiency of recombinantly expressed deletion mutants of the murine AHSG domain D1 revealed that the structure built up by the amino acids 15-70, i.e. the aminoterminal alpha-helix and the first two strands of the beta sheet, contain the complete inhibitory potential. Futhermore, functional testings evidenced that no other cystatin-like domain inhibits the precipitation with a comparable effiency because their modelled structures lack a comparable density of acidic residues on the beta-sheet. Tests of the remaining inhibitory potential of sera from dialysis-patients and Ahsg deficient mice that both already had developed severe ectopic calcifications in various organs, proved the importance of AHSG as an essential soluble inhibitor of calcium phosphate precipitation. Considering the solubility product of hydroxyapatite and the maximal amount of AHSG bound calcium, the inhibition cannot be explained by a reduction of the ion product. In fact, the inhibition relies on the adsorption of AHSG on calcium phosphate surfaces. This was demonstrated firstly by a change in morphology of the precipitate induced by AHSG as observed by scanning electron microscopy and secondly by visualisation of soluble AHSG-calcium phosphate aggregates by transmission electron microscopy. Obviously, AHSG binds to nascent calcium phosphate aggregates with a high affinity and thus prevents their further growth by a reduction of the diffusion rate of calcium and phosphate ions to the surface.
info:eu-repo/classification/ddc/540, Chemie
Networked Digital Library of Theses and Dissertations
Publikationsserver der RWTH Aachen University