The role of heparan sulfate in cathepsin K biology

破骨细胞活动过度是许多骨骼疾病的基础，如骨质疏松症、牙周炎、类风湿 关节炎和佩吉特氏病。骨吸收需要降解有机骨基质，其主要特征是 成分为I型胶原蛋白。为了降解骨基质，破骨细胞分泌组织蛋白酶K(CTSK)，一种有效的 胶原酶在酸性pH条件下作用最好。CTSK基因缺失的小鼠表现出严重的骨化表型 尽管破骨细胞分化正常，但这有力地支持了CTSK在骨骼中的基本作用 再吸收。CTSK是一种高度正电的蛋白质，已知能结合带负电荷的糖胺多聚糖， 其中，它与硫酸乙酰肝素(HS)结合最强烈。广泛分布在细胞表面和细胞内 细胞外基质，可想而知CTSK与HS之间的高亲和力相互作用在CTSK中起一定作用 生物学。然而，HS如何调节CTSK的生物学功能仍是完全未知的。这个 这一关键的知识鸿沟的持续存在阻碍了我们对CTSK的更全面的了解 生物学，可能还来自于发挥靶向CTSK的治疗潜力。我们的中心假设是 硫酸乙酰肝素调节CTSK的活性和定位，CTSK的活性可以 通过靶向其HS结合位点来操纵。在实验室产生的强大初步数据的指导下，我们的 假设将通过追求以下三个具体目标来检验。目标1：确定结构 CTSK-HS交互的详细信息。我们将采用互补的生物物理/生化方法，包括 X射线结晶学、小角X射线散射和HS寡糖微阵列 CTSK-HS相互作用的结构基础。目的2：确定CTSK-HS相互作用的生物学作用。通过 利用新产生的带有HS结合缺陷CTSK(CTSK∆HS)的敲入小鼠品系，我们将尝试 了解CTSK-HS相互作用中断的生理后果。目的3：对CTSK的抑制 结构上定义的HS寡糖。我们将检测CTSK结合HS的抗吸收活性 低聚糖在一些小鼠骨重塑模型中的作用。通过研究CTSK-HS的相互作用 作为体外和体内方法的结合，我们的贡献将是重大的，因为我们将识别 HS调节CTSK生物学功能的多种机制。如果没有这种批判性知识， 我们对CTSK生物学的理解永远不会完整。我们的调查结果也可能会有所帮助 设计新的方法来操纵这种高度重要的蛋白酶的活性。尤其是因为HS 具有选择性抑制CTSK胶原酶活性的独特性质，而不影响 其正常的多肽酶活性，我们的研究可能有助于开发一种理想的CTSK抑制剂来治疗许多骨骼 疾病。

Excessive osteoclast activity underlies many bone diseases such as osteoporosis, periodontitis, rheumatoid arthritis and Paget’s disease. Bone resorption requires degradation of organic bone matrix, whose main component is type I collagen. To degrade bone matrix, osteoclasts secrete cathepsin K (CtsK), a potent collagenase that functions optimally under acidic pH. CtsK-null mice display severe osteopetrotic phenotype despite normal differentiation of osteoclasts, which strongly supports the essential role of CtsK in bone resorption. A highly positively charged protein, CtsK is known to bind negatively charged glycosaminoglycans, among which it binds heparan sulfate (HS) most avidly. Widely distributed at the cell surface and in the extracellular matrix, it is conceivable that the high-affinity interaction between CtsK and HS plays a role in CtsK biology. However, how HS regulates the biological functions of CtsK remains completely unknown. The persistence of this critical knowledge gap prevents us from gaining a more complete understanding of CtsK biology, and likely also from fulfilling the therapeutic potential of targeting CtsK. Our central hypothesis is that heparan sulfate regulates the activity and localization of CtsK, and that CtsK activity can be manipulated by targeting its HS-binding site. Guided by strong preliminary data generated in the lab, our hypothesis will be tested by pursuing the following three specific aims. Aim 1: Determine the structural details of CtsK-HS interaction. We will employ complementary biophysical/biochemical methods including X-ray crystallography, small-angle X-ray scattering and HS oligosaccharide microarray to understand the structural basis of CtsK-HS interaction. Aim 2: Determine the biological roles of CtsK-HS interaction. By utilizing a newly generated knock-in mouse strain bearing an HS-binding deficient CtsK (CtsK∆HS), we will try to understand the physiological consequence of disrupted CtsK-HS interaction. Aim 3: Inhibition of CtsK with structurally defined HS oligosaccharides. We will examine the antiresorptive activity of CtsK-binding HS oligosaccharides in a number of murine models of bone remodeling. By investigating CtsK-HS interaction using a combination of in vitro and in vivo methods, our contribution will be significant because we will identify multiple mechanisms by which HS regulates the biological functions of CtsK. Without this critical knowledge, our understanding of CtsK biology will never be complete. The outcome of our investigation could also help design novel ways of manipulating the activity of this highly important protease. In particular, because HS possesses the unique property of selectively inhibiting the collagenase activity of CtsK without compromising its normal peptidase activity, our investigation might help develop an ideal CtsK inhibitor for treating many bone diseases.