骨唾液酸蛋白糖基化修饰对骨形成的影响及其作用机制的研究

Bone is renewed to maintain its strength and mineral homeostasis by remodeling which is composed of a tightly coupled group of osteoclasts and osteoblasts to carry out resorption of old bone and formation of new bone. Disruption of this homeostasis leads to metabolic bone diseases such as osteoporosis and bone metastasis of tumors and is a major health risk factor in post menopausal women and in men above 50 years old. Homeostasis of bone resorption and formation has drawn tremendous research attentions over the last couple of decades. However, cellular and molecular mechanisms underlying its regulation are still not well understood. Bone-forming osteoblasts are responsible for the secretion of a distinct organic extracellular matrix (ECM) that will ultimately become mineralized and lead to bone formation. Bone sialoprotein (BSP) is an abundant non-collagenous glycophosphoproteins in the ECM of bone. In addition to calcification of newly synthesized organic matrix, BSP also promotes cell proliferation and expression of osteoblastic phenotypes, suggesting a role in cellular signaling. Our recent study demonstrated that cell surface α2-3 sialic acid plays important role in osteogenesis using a cell culture model of osteoblast differentiation. Accordingly, we hypothesize that glycosylation is necessary for BSP effects on osteogenesis. In this proposal, we will examine for the first time the specific glycosylation sites (N-glycosylation and O-glycosylation) and carbohydrate linkages using the state of the art mass spectrometry. We will then study their specific effects in regulating osteogenesis and signaling pathways which are important for osteocyte differentiation. To explore clinical importance of our findings in vitro, we will then examine whether BSP levels and its glycosylation play important roles in bone metastasis in breast cancer patients. BSP levels and glycosylation patterns in breast cancer patients with or without bone metastasis will be measured. Molecular mechanisms leading to the observed regulation will be further explored using cellular as well as commonly used bone metastasis animal models. Completion of this study will likely provide novel knowledge on molecular mechanisms regulating osteogenesis and bone metastasis in breast cancer patients. This study will also help evaluate the feasibility of using BSP and its glycosylation linkages as pharmaceutical targets to treat metabolic bone disorders and bone metastasis in cancers.

骨唾液酸蛋白(Bone Sialoprotein,BSP)是一种富含末端唾液酸糖链结构的高度磷酸化和高度糖基化的细胞外基质蛋白，此蛋白在生长和修复的新骨及某些有骨转移倾向的肿瘤中高表达。申请人在前期研究中率先证实BSP在体内具有促成骨作用，其他体外研究提示此蛋白促成骨作用并非由磷酸化修饰所致。由此，申请人假设BSP的糖基化修饰在骨形成中起关键作用。为论证此假设，本课题将利用申请人已掌握的BSP提取技术、糖组学和质谱分析技术。从小牛骨中获取和制备完整糖链、去部分糖链及无糖链的BSP，分析这些具有不同程度糖基化的BSP在前成骨细胞培养、颅骨缺损模型中的促成骨作用。并在此基础上探索BSP糖基化激发的特异性信号通路对骨形成的影响。本课题的完成将明确BSP糖基化修饰在骨形成中的作用及机制，并为开发以BSP为靶蛋白的临床治疗骨代谢疾病提供实验基础和理论依据。

在骨形成过程中骨形成蛋白、骨唾液酸蛋白(Bone Sialoprotein, BSP)、骨桥蛋白、胶原蛋白等起着重要的作用，而这些蛋白质都属于糖蛋白，特别是BSP，目前己将其作为一种新的骨生长因子进行研究。BSP存在多个O-糖基化位点和潜在的N-糖基化位点，并具有O-糖链和结构较复杂的N-糖链，以维持其构象稳定，但其糖链结构的功能尚属未知。我们在掌握提取和鉴定天然BSP的基础上，用不同的糖苷酶消化rhiBSP，并利用生物素标记的凝集素结合，用PE标记的亲和素作为二抗结合凝集素上携带的生物素，Western Blot分析显示rhiBSP可以被不同程度地糖基化，同时利用广谱蛋白酶对rhiBSP酶解得到的糖肽富集后采用HCD-pd-ETD 的模式进行二级质谱鉴定，并利用Byonic软件和57种人血清中的N糖链组成的数据库，在所得质谱数据中鉴定到了四个N糖基化位点（N104, N177, N182, N190）处28种不同糖型的糖肽，这是第一次在实验中证实了rhiBSP中存在高度唾液酸化的N糖链，不同的脱糖基化样品的唾液酸定量分析研究也表明，N、O 糖链均存在唾液酸化，这与之前报道的模型研究结果一致，这些结果进一步证实BSP是一个含N-糖链、O-糖链及末端唾液酸结构的糖基化蛋白。由此，我们在建立MC3T3-E1 Subclone 14体外成骨细胞培养模型中，ALP、von Kossa染色证实BSP能促进成骨和骨细胞的形成，而加入各种糖基化修饰的BSP能影响体外成骨细胞或骨细胞的形成，特别是去除BSP末端唾液酸后，western-blot检测发现去末端唾液酸化BSP能明显影响骨矿化指标OPG、VDR及p-Erk/t-Erk的表达，加入Erk抑制剂PD0325901，发现其能抑制VDR的表达，而对OPG和RANKL无明显影响。以通讯作者发表相关SCI论文两篇。