Choi J Y, van Wijnen A J, Aslam F, Leszyk J D, Stein J L, Stein G S, Lian J B, Penman S
Department of Cell Biology and Cancer Center, and Protein Chemistry Facility, University of Massachusetts Medical Center, Worcester, MA 01655, USA.
J Cell Sci. 1998 Oct;111 ( Pt 20):3035-43. doi: 10.1242/jcs.111.20.3035.
The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.
随着成骨细胞从增殖的前成骨细胞成熟为嵌入矿化基质中的骨细胞，核基质的蛋白质组成发生显著变化。这些基质蛋白是成骨细胞分化过程中发育阶段特异性基因表达的结果。为了分离出骨表型特有的核基质蛋白，我们在两个不同阶段，即增殖期（第3天）和分化期（第18天，矿化），通过高分辨率二维凝胶电泳分析了大鼠颅骨成骨细胞培养物的核基质制剂。我们鉴定出一种蛋白质（14 kDa；pI 5.0），该蛋白质仅在分化的成骨细胞核基质中可检测到。通过质谱分析和微量测序，该蛋白质被鉴定为β-半乳糖苷结合蛋白半乳凝素-1。用半乳凝素-1抗体对核基质制剂进行免疫荧光染色以及对亚细胞组分进行蛋白质印迹分析均证实，由于差异性保留，半乳凝素-1仅与分化的成骨细胞核基质相关。在整个成骨细胞分化过程中都存在半乳凝素-1蛋白和mRNA。在增殖和成骨细胞中，半乳凝素-1都存在于细胞质和细胞核组分中。然而，它唯一稳定的结合是与分化的成骨细胞核基质结合；但是，在增殖的成骨细胞中，半乳凝素-1不会保留在核基质中。综上所述，我们的结果表明，半乳凝素-1与核基质的发育关联反映了成骨细胞分化过程中半乳凝素-1在核内的差异性结合。
