Analysis of Receptor for Bone Resorption Suppressor Caldecrin

急性胰腺炎患者血清钙降低（低钙血症）。我们从胰腺中分离纯化了一种血清钙降低因子，命名为钙调蛋白，它是一种胰凝乳蛋白酶型丝氨酸蛋白酶。由于钙调蛋白具有抑制骨吸收的作用，本课题研究了钙调蛋白抑制骨吸收的结构域及其受体。将Caldecnn片段与组氨酸标记的硫氧还蛋白融合以产生二硫键。将含有钙调蛋白片段的表达载体转化大肠杆菌，IPTG诱导表达，亲和层析柱纯化。采用兔破骨细胞挖骨陷窝法测定骨吸收活性。钙调蛋白由2个β-桶状结构组成。当钙调蛋白被分成2部分时，发现两部分都具有骨吸收抑制活性。抑制活性位于前桶结构的后半部分。破坏后一桶结构的二硫桥不改变抑制活性。钙调蛋白抑制纯化的兔破骨细胞的骨吸收，提示兔破骨细胞上存在钙调蛋白受体。因此，我们使用SMART oligo和oligo（dT）引物，通过PCR从10^4个破骨细胞中构建了兔破骨细胞cDNA文库。将具有表达载体的重组大肠杆菌分成亚池。因此，我们可以利用抗硫氧还蛋白抗体建立兔破骨细胞受体基因的克隆系统。

A reduction of serum calcium (hypocalcemia) occurs in patients with acute pancreatitis. We purified the serum calcium-decreasing factor from pancreas and named caldecrin, which found to be a chymotrypsin-type serine protease. Science the caldecrin suppressed a bone resorption, we studied to determine a suppressive domains for the bone resorption and its receptors. Caldecnn fragments were fused to Hislidine-tagged thioredoxin to produce disulfide bridge. Expression vector with caldecrin fragment were transformed into E.Coli, induced with IPTG, and purified by affinity chelating column. Bone resorption activity was measured by a method of excavated bone resorption pit formation by rabbit osteoclasts. The caldecrin is composed of 2 beta-barrel like structures. When the caldecrin was divided into 2 parts, the suppression activities for bone resorption were found to be both parts. The suppressive activity was localized on a latter half part of the former barrel structure. Disruption of disulFide bridge of the latter barrel structure was not changed the suppressive activity. Caldecrin suppressed the bone resorption of purified rabbit osteoclasts, suggesting the existence of caldecrin receptors on the rabbit osteoclasts. Thus, we made rabbit osteoclast cDNA library from lO^4 osteoclasts using PCR with SMART oligo and oligo (dT) primers. Recombinant E.Colis with expression vector were divided into subpools. Thus we could established the cloning system for rabbit osteoclst receptor genes using anti-thioredoxin antibody.