Wijeratne Sithara S, Martinez Jerahme R, Grindel Brian J, Frey Eric W, Li Jingqiang, Wang Liyun, Farach-Carson Mary C, Kiang Ching-Hwa
Department of Physics and Astronomy, Rice University, Houston, TX 77005, USA.
Department of BioSciences, Rice University, Houston, TX 77005, USA.
Matrix Biol. 2016 Mar;50:27-38. doi: 10.1016/j.matbio.2015.11.001. Epub 2015 Nov 4.
Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170±20 nm and a diameter of 2-4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young's modulus of 71 MPa. We conclude that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.
基底膜聚糖/HSPG2是一种大型单体硫酸乙酰肝素蛋白聚糖（HSPG），是皮质骨腔管系统（LCS）的关键组成部分，它是围绕骨细胞突起的机械传感细胞周基质（PCM）的一部分，并作为将骨细胞胞体连接到骨基质的拴系元件。在骨细胞胞体周围的细胞周空间内，基底膜聚糖会受到生理流体流动阻力，并以此作为传感器将外部刺激传递到骨细胞膜。我们之前表明，基底膜聚糖分泌减少会改变PCM纤维组成，并干扰体内骨骼对机械负荷的反应。为了验证我们的假设，即基底膜聚糖核心蛋白在生理负荷条件下能够承受拉力而不发生解折叠，我们使用原子力显微镜（AFM）以纳米级分辨率捕获基底膜聚糖单体的图像，并进行单分子力测量（SMFM）。我们发现，纯化的全长人基底膜聚糖的核心蛋白大小适合跨越LCS的细胞周空间，测得的端到端长度为170±20 nm，直径为2 - 4 nm。拉力显示出一个强大的蛋白核心，它能够承受超过100 pN的拉力，远超过估计施加在单个骨细胞拴系上阻力。用可扩展蠕虫状链模型进行数据拟合表明，基底膜聚糖蛋白核心的平均弹性常数为890 pN，相应的杨氏模量为71 MPa。我们得出结论，基底膜聚糖具有使其能够在LCS中充当强而有弹性的拴系的物理特性。
