Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin).
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.
Tomomura
A
A
Department of Biochemistry, Meikai University School of Dentistry, 1-1 Keyakidai, Saitama 350-0283, Sakado, Japan. atomomu@dent.meikai.ac.jp
Yamada
H
H
Fujimoto
K
K
Inaba
A
A
Katoh
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001862:Bone Resorption; D002118:Calcium / Q000097:blood; D002918:Chymotrypsin / Q000737:chemistry; D004792:Enzyme Precursors / Q000737:chemistry / Q000494:pharmacology; D009154:Mutation; D010010:Osteoclasts / Q000187:drug effects*; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010664:Phenylmethylsulfonyl Fluoride; D012697:Serine Endopeptidases; C074385:caldecrin; D002918:Chymotrypsin; C117569:chymotrypsin C; D002118:Calcium