Kojima T, Miyaishi O, Saga S, Ishiguro N, Tsutsui Y, Iwata H
Department of Orthopedic Surgery, Nagoya University School of Medicine, Japan.
J Pathol. 1998 Feb;184(2):212-8. doi: 10.1002/(SICI)1096-9896(199802)184:2<212::AID-PATH996>3.0.CO;2-Z.
Various mutations of genes encoding type I procollagen chains have been linked to osteogenesis imperfecta (OI). The mutations yield abnormal procollagen molecules that fold improperly. HSP 47, a stress-inducible protein localized to the endoplasmic reticulum (ER) of collagen-producing cells, may participate in collagen processing as a procollagen-specific molecular chaperone. The intracellular transport of abnormal procollagen molecules and the expression of HSP 47 have been studied in fibroblasts from a patient with OI. Normal and OI fibroblasts cultured with or without ascorbate were analysed by immunofluorescent double labelling with monoclonal antibodies to C-propeptide of type I procollagen and HSP 47, as observed by confocal microscopy. Procollagen and HSP 47 were also quantified by immunoprecipitation of normal and OI fibroblasts radiolabelled with 35S-methionine. By confocal microscopy, procollagen molecules were retained in the ER of both fibroblast types cultured in the absence of ascorbate, and were co-localized with HSP 47. In normal fibroblasts, 2 h after the addition of ascorbate, most of the procollagen had disappeared from the cells, while in OI fibroblasts, abnormal procollagen molecules and HSP 47 were still retained in the ER. By immunoprecipitation, procollagen was negligible in normal fibroblasts cultured with ascorbate; much larger amounts of procollagen were immunoprecipitated from OI fibroblasts despite ascorbate. Increased HSP 47 in OI fibroblasts was demonstrated by immunoprecipitation with a specific monoclonal antibody. These results suggest the increase in HSP 47 in the ER of OI fibroblasts is related to its collagen-specific chaperone function.
编码I型前胶原链的基因发生的各种突变已与成骨不全症（OI）相关联。这些突变产生折叠不正确的异常前胶原分子。HSP 47是一种定位于产生胶原蛋白细胞内质网（ER）的应激诱导蛋白，可能作为一种前胶原特异性分子伴侣参与胶原蛋白的加工过程。在一名OI患者的成纤维细胞中，对异常前胶原分子的细胞内运输和HSP 47的表达进行了研究。用针对I型前胶原C-前肽和HSP 47的单克隆抗体进行免疫荧光双重标记，对在有或没有抗坏血酸的情况下培养的正常和成骨不全症成纤维细胞进行分析，通过共聚焦显微镜观察。前胶原和HSP 47也通过对用35S-甲硫氨酸进行放射性标记的正常和成骨不全症成纤维细胞进行免疫沉淀来定量。通过共聚焦显微镜观察，在没有抗坏血酸的情况下培养的两种成纤维细胞类型中，前胶原分子都保留在内质网中，并与HSP 47共定位。在正常成纤维细胞中，添加抗坏血酸2小时后，大多数前胶原已从细胞中消失，而在成骨不全症成纤维细胞中，异常前胶原分子和HSP 47仍保留在内质网中。通过免疫沉淀，在用抗坏血酸培养的正常成纤维细胞中前胶原可以忽略不计；尽管有抗坏血酸，从成骨不全症成纤维细胞中免疫沉淀出的前胶原量要大得多。用特异性单克隆抗体进行免疫沉淀证明成骨不全症成纤维细胞中HSP 47增加。这些结果表明，成骨不全症成纤维细胞内质网中HSP 47的增加与其胶原特异性伴侣功能有关。
