Multiple molecular forms of pyridinoline crosslinks generated by the action of cathepsin B on bone collagen : Influence of high calcium concentrations

الملخص EN

We investigated in vitro the ability of cathepsin B, a lysosomal cysteine proteinases, to generate multiple molecular forms of pyridinoline crosslinks from insoluble bone type I collagen, and we studied the effects ofvarious concentrations of divalent ions such as calcium (Ca2+) and magnesium (Mg2+) on this process.

Our results have shown that the addition of either Ca2+ or Mg2+ greatly enhanced the release of pyridinoline crosslinks, the greatest effects being obtained at 50-75 mM.

By dialysis and gel filtration chromatography methods, we attempted to separate the different molecular species of peptides containing pyridinoline crosslinks resulting from the digestion of bone with catepsin B.

In a Ca2+ and Mg2+ free buffer, the molecular weight of approximately 80 % of the peptide fragments containing crosslinks was under 3500 Da.

Addition of MgCl2 (40 mM) did not modified significantly this repartition, while CaCl2 changed it dramatically.

Indeed, in presence of various concentrations of CaCl2 (10-40 mM), the proteolytic balance of the enzyme was shifted toward the release of peptide-bound crosslinks fragments with higher size (MW > 3500 Da).

These data could suggest that locally elevated concentrations of Ca2+, such as those seen in the bone-resorbing microenvironment, could modify the proteolysis of bone collagen mediated by lysosomal cysteines proteinases.