Department of Orthodontics, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, 464-8650, Japan.
Department of Biochemistry, School of Dentistry, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, 464-8650, Japan.
Biochem Biophys Res Commun. 2022 Dec 20;635:203-209. doi: 10.1016/j.bbrc.2022.10.042. Epub 2022 Oct 14.
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 with Gaussia luciferase (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.
首次使用视频速率生物发光成像方法可视化活成骨细胞分泌的骨钙素和骨形态发生蛋白 2（BMP2）。在成骨细胞 MC3T3-E1 细胞中表达的与 Gaussia 荧光素（GLase）融合的小鼠骨钙素和 BMP2（OC-GLase 和 BMP2-GLase）分别被正确加工和分泌。在胞吐分泌的视频图像中，OC-GLase 和 BMP2-GLase 的发光斑点分别迅速和逐渐消失，表明这些蛋白质的扩散方式不同。值得注意的是，BMP2 的缺失突变体（Δ3BMP2-GLase）缺失了 N 端与肝素硫酸盐结合的三个碱性氨基酸残基，显示出迅速消失的发光斑点。在我们的成像条件下，Δ3BMP2-GLase 斑点的发光半衰期（1.61±0.20 s）与 OC-GLase（1.22±0.14 s）相似，但与 BMP2-GLase（4.31±0.41 s）不同。这些结果表明，与骨钙素不同，BMP2 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可用于研究分泌蛋白在胞吐作用中的扩散特性。
