Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging
Visualization of osteocalcin and bone morphogenetic protein 2 (BMP2) secretion from osteoblastic cells by bioluminescence imaging
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DOI：
10.1016/j.bbrc.2022.10.042
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发表时间：
2022-10-21
影响因子：
3.1
通讯作者：
Suzuki，Takahiro
中科院分区：
文献类型：
--
作者：
Sugimoto，Honami;Fukuda，Shinji;Suzuki，Takahiro
利用生物荧光成像技术，首次观察到成骨细胞分泌的骨钙素和骨形态发生蛋白2（BMP 2）。在成骨细胞MC 3 T3-E1中表达的小鼠骨钙素和BMP 2的高斯荧光素酶（GLase）融合蛋白（分别为OC-GLase和BMP 2-GLase）被正确加工和分泌。在细胞外分泌的视频图像中，OC-GLase和BMP 2-GLase的发光斑点分别快速和逐渐消失，表明这两种蛋白质在扩散中的不同方式。值得注意的是，BMP 2的缺失突变体（Δ 3BMP 2-GLase）在N-末端区域中缺少用于结合硫酸乙酰肝素的三个碱性氨基酸残基，显示出快速消失的发光斑点。在我们的成像条件下，Δ 3BMP 2-GLase斑点的发光半衰期（1.61 ± 0.20 s）与OC-GLase斑点的发光半衰期（1.22 ± 0.14 s）相似，但与BMP 2-GLase斑点的发光半衰期（4.31 ± 0.41 s）不同。这些结果表明，与骨钙素相反，BMP 2从细胞中的扩散在胞吐后缓慢发生。因此，我们的生物发光成像方法是有用的，以研究在胞吐分泌蛋白质的扩散特性。
The secretions of osteocalcin and bone morphogenetic protein 2 (BMP2) from living osteoblastic cells were visualized for the first time using a method of video-rate bioluminescence imaging. The fusion proteins withGaussialuciferase (GLase) for mouse osteocalcin and BMP2 (OC-GLase and BMP2-GLase, respectively) expressed in osteoblastic MC3T3-E1 cells were correctly processed and secreted. In the video images of exocytotic secretion, the luminescence spots of OC-GLase and BMP2-GLase disappeared rapidly and gradually, respectively, indicating different manners of these proteins in diffusion. Notably, a deletion mutant of BMP2 (Δ3BMP2-GLase) lacking three basic amino acid residues in the N-terminal region for binding to heparan sulfate showed rapidly disappearing luminescence spots. In our imaging conditions, the half-life of luminescence for the spots of Δ3BMP2-GLase (1.61 ± 0.20 s) was similar to that of OC-GLase (1.22 ± 0.14 s) but not to that of BMP2-GLase (4.31 ± 0.41 s). These results suggest that, in contrast to osteocalcin, the diffusion of BMP2 from cells occurred slowly after exocytosis. Thus, our bioluminescence imaging method is useful to study the diffusion properties of secreted proteins in exocytosis.