Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin)

Abstract

We previously reported on the serum calcium-decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315–30321]. To address the mechanism of this serum calcium-decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone-resorbing activity. Wild-type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose-dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease-deficient mutant caldecrins. Recombinant procaldecrin (−13–239), and its fragments (−13–125), (1–111), (1–46), (47–111), and (126–239) were expressed as His-tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (−13–239) and fragment (−13–125) did not affect the suppressive activity, whereas fragments (1–111) and (126–239) did suppress the bone resorption. The bone-resorbing activity was also suppressed by fragment (47–111), not by fragment (1–46). Overlapping fragments (47–62), (47–79), (47–98), (56–111), (71–111), and (85–111) were compared for their suppressive activity. The fragments (47–62) and (85–111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71–79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71–79) is responsible for the suppression of bone resorption by caldecrin.