Division of Bone Diseases, Geneva University Hospital, Faculty of Medicine, Geneva, Switzerland.
Calcif Tissue Int. 2017 Nov;101(5):501-509. doi: 10.1007/s00223-017-0302-3. Epub 2017 Jul 19.
Periostin is a matricellular protein mainly expressed by periosteal cells and osteocytes in bone, but is also present in several other tissues. Available immunoassays use antibodies of unclear specificity. The aim of the study was to develop a bone-specific periostin ELISA based on the detection of fragments generated by the osteoclastic and osteocytic protease cathepsin K. In vitro digestion of human recombinant intact periostin by cathepsin K leads to the generation of multiple fragments. Using LS-MS/MS, it was found that the GSLQPIIK peptide was the most efficiently and abundantly generated periostin fragment. A rabbit polyclonal antibody directed against the synthetic GSLQPIIK sequence was produced. Immunohistochemistry experiments of the tibia showed that the GSLQPIIK fragments localized at the periosteal surface and within the osteocytes. Using the same antibody, we developed an ELISA for the measurement of GSLQPIIK in the serum. This ELISA demonstrated intra- and interassay variability below 14% with a sensitivity allowing accurate determinations in the serum of healthy individuals. Serum GSLQPIIK was measured in 160 healthy postmenopausal women (mean age 65 year) participating in the Geneva Retiree Cohort. Serum GSLQPIIK levels did not correlate with total periostin, hip BMD, and the bone markers PINP and CTX. However, GSLQPIIK was negatively correlated (p values ranging from 0.007 to 0.03) with Hr-pQCT measures of tibia and radius cortical bone, but not with trabecular parameters. We have developed the first assay for the detection of periostin fragments generated by cathepsin K. Because serum levels of this new marker significantly correlated with cortical bone measurements in postmenopausal women, it may prove to be useful for the clinical investigation of patients with osteoporosis.
骨膜蛋白是一种细胞外基质蛋白，主要由骨膜细胞和骨细胞表达，但也存在于其他几种组织中。现有的免疫检测方法使用的抗体特异性不明确。本研究旨在开发一种基于骨组织特异性蛋白酶半胱氨酸蛋白酶 K 对骨膜蛋白的检测的骨膜蛋白 ELISA。体外实验中，骨膜蛋白经半胱氨酸蛋白酶 K 消化后可产生多个片段。利用 LS-MS/MS 技术，发现 GSLQPIIK 肽是生成效率最高、含量最丰富的骨膜蛋白片段。我们制备了针对该合成肽段的兔多克隆抗体。对胫骨的免疫组织化学实验显示，GSLQPIIK 片段定位于骨膜表面和骨细胞内。我们利用该抗体开发了用于检测血清中 GSLQPIIK 的 ELISA。该 ELISA 实验的批内和批间变异性均低于 14%，检测灵敏度足以准确测定健康个体的血清样本。我们对参加日内瓦退休人员队列研究的 160 名绝经后健康女性（平均年龄 65 岁）的血清 GSLQPIIK 进行了检测。血清 GSLQPIIK 水平与总骨膜蛋白、髋部 BMD 以及骨形成标志物 PINP 和 CTX 均无相关性。然而，GSLQPIIK 与 HR-pQCT 测量的胫骨和桡骨皮质骨呈负相关（p 值范围为 0.007 至 0.03），但与松质骨参数无关。我们开发了检测半胱氨酸蛋白酶 K 生成的骨膜蛋白片段的首个检测方法。由于该新标志物的血清水平与绝经后女性的皮质骨测量值显著相关，因此它可能对骨质疏松症患者的临床研究具有重要价值。
