Innes-Gold Sarah N, Berezney John P, Saleh Omar A
Materials Department, University of California, Santa Barbara, Santa Barbara, California.
Materials Department, University of California, Santa Barbara, Santa Barbara, California; Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California.
Biophys J. 2020 Oct 6;119(7):1351-1358. doi: 10.1016/j.bpj.2020.08.016. Epub 2020 Aug 20.
Large bottlebrush complexes formed from the polysaccharide hyaluronan (HA) and the proteoglycan aggrecan contribute to cartilage compression resistance and are necessary for healthy joint function. A variety of mechanical forces act on these complexes in the cartilage extracellular matrix, motivating the need for a quantitative description that links their structure and mechanical response. Studies using electron microscopy have imaged the HA-aggrecan brush but require adsorption to a surface, dramatically altering the complex from its native conformation. We use magnetic tweezers force spectroscopy to measure changes in extension and mechanical response of an HA chain as aggrecan monomers bind and form a bottlebrush. This technique directly measures changes undergone by a single complex with time and under varying solution conditions. Upon addition of aggrecan, we find a large swelling effect manifests when the HA chain is under very low external tension (i.e., stretching forces less than ∼1 pN). We use models of force-extension behavior to show that repulsion between the aggrecans induces an internal tension in the HA chain. Through reference to theories of bottlebrush polymer behavior, we demonstrate that the experimental values of internal tension are consistent with a polydisperse aggrecan population, likely caused by varying degrees of glycosylation. By enzymatically deglycosylating the aggrecan, we show that aggrecan glycosylation is the structural feature that causes HA stiffening. We then construct a simple stochastic binding model to show that variable glycosylation leads to a wide distribution of internal tensions in HA, causing variations in the mechanics at much longer length scales. Our results provide a mechanistic picture of how flexibility and size of HA and aggrecan lead to the brush architecture and mechanical properties of this important component of cartilage.
由多糖透明质酸（HA）和蛋白聚糖聚集蛋白聚糖形成的大型瓶刷状复合物有助于软骨抗压，是关节健康功能所必需的。多种机械力作用于软骨细胞外基质中的这些复合物，因此需要对其结构和力学响应进行定量描述。使用电子显微镜的研究对HA-聚集蛋白聚糖刷进行了成像，但需要吸附到表面，这会使复合物从其天然构象发生显著改变。我们使用磁镊力谱来测量随着聚集蛋白聚糖单体结合并形成瓶刷时HA链的伸长变化和力学响应。该技术直接测量单个复合物在不同溶液条件下随时间发生的变化。加入聚集蛋白聚糖后，我们发现当HA链处于非常低的外部张力下（即拉伸力小于约1皮牛）时会出现很大的膨胀效应。我们使用力-伸长行为模型表明，聚集蛋白聚糖之间的排斥会在HA链中诱导内部张力。通过参考瓶刷聚合物行为理论，我们证明内部张力的实验值与多分散的聚集蛋白聚糖群体一致，这可能是由不同程度的糖基化引起的。通过对聚集蛋白聚糖进行酶促去糖基化，我们表明聚集蛋白聚糖糖基化是导致HA变硬的结构特征。然后我们构建了一个简单的随机结合模型，以表明可变糖基化会导致HA内部张力的广泛分布，从而在更长的长度尺度上引起力学变化。我们研究结果提供了一个机制图，说明HA和聚集蛋白聚糖的柔韧性和大小如何导致这种软骨重要组成部分的刷状结构和力学性能。
