Molecular Cloning and Expression of Serum Calcium- decreasing Factor (Caldecrin)

Abstract

We previously reported on the purification of a serum calcium-decreasing factor, referred to as caldecrin, from porcine pancreas, that is thought to be a serine protease (Tomomura, A., Fukushige, T., Noda, T., Noikura, T., and Saheki, T. (1992) FEBS Lett. 301, 277-281). In the present study, we purified caldecrin from rat pancreas and determined its primary structure by cDNA cloning. The predicted caldecrin protein is presumed to be synthesized as a preproenzyme of 268 amino acids with a signal peptide of 16 amino acids and an activation peptide of 13 amino acids, and is, with the exception of a central region, almost identical to the reported rat pancreatic elastase IV sequence. The caldecrin gene is selectively expressed in the pancreas, as judged by Northern blot analysis. After expression in BMT-10 cells, immunoreactive caldecrin was found in the culture supernatant, and it inhibited the parathyroid hormone-stimulated 45 Ca release from cultured fetal long bones. Catalytic site mutants were synthesized in a baculovirus system, and recombinant mutants also decreased the serum calcium level of mice. These data implicate caldecrin, a protease closely related to elastase IV, in the regulation of blood calcium levels.

Molecular cloning and expression of human caldecrin

“…The amino acid sequence of fragby stepwise increases in the phosphate buffer concentration up to 0.5 M. Caldecrin in each fraction was assayed for proteolytic activity ments derived from purified rat caldecrin was identical to the after trypsin activation, and caldecrin protein was detected by Wesamino acid sequence deduced from rat caldecrin cDNA but tern blotting using anti-porcine caldecrin antibody. different from that of elastase IV, especially in the central region [4].…”

“…quence of purified porcine caldecrin were reported previously [4]. The arrowheads indicate the proteolytic cleavage sites.…”

“…The serum calcium-decreasing activity (SCDA) was measured as described previously [4]. Recombinant caldecrin was activated at 24°C for 30 min with trypsin at 1/50th of the protein amount of 2.…”

“…Sf9 cells were transfected with the recombinant virus and cultured rat caldecrin cDNA [2][3][4]. The primary structure of the rat for 3 days.…”

“…Serum calcium concentration was measured by the o-cresolphthalein complexson method [7]. The amino acid sequences deduced from human caldecrins HC1 and HC2 were compared with those of rat caldecrin [4], porcine elastase I [8], rat elastase I [9], porcine elastase II [10], rat elastase II [9], human elastase IIA and IIB [10], and human elastase IlIA and IIIB [11].…”