卵泡刺激素生物活性相关的糖基化研究

Follicle-stimulating hormone (FSH) is a hormonal therapeutic glycoprotein and is widely used for in vitro fertilization. Glycans significantly influence the biological activity of FSH, including dimer assembly, signal transduction, endocrine secretion, the threshold of ovarian follicles and follicular growth. Glycosylation is the critical quality attribution (CQA) of FSH, but the high heterogeneity and variety of FSH glycosylation hamper quality improvement and the development of novel preparations. Moreover, two classes of FSH-containing pharmaceutical preparations currently exist; those manufactured using recombinant DNA technology (rhFSH) and those derived from the urine of postmenopausal women (uhFSH). Due to the differences of glycosylation between rhFSH and uhFSH, their half-life and effects in clinical applications vary. Using various glycosylation analysis and bioassay technologies, our research identifies glycan differences and its relationship with biological activities of rhFSH/uhFSH to reveal the critical glycans. Taking advantage of chemoenzymatic synthesis, glyco-remodeled FSH is prepared for further study of critical glycans. Furthermore, glycosylated homogeneous FSH with higher activity is expressed in glycoengineering cell lines. The aim of this study is to improve hFSH quality and promote the development of novel preparations. This work also provides new thoughts and strategies of glycosylation study for therapeutic glycoproteins.

卵泡刺激素(FSH)是一种糖蛋白激素药物，广泛应用于辅助生殖。研究表明卵泡刺激素的糖基化显著影响其生物活性，包括亚基整合、信号转导、性激素分泌及卵泡大小和生长。虽然糖基化是FSH的关键质量属性，但由于其高度不均一和多样性，阻碍FSH质量提高和新型制剂研发。此外，卵泡刺激素有两种来源：重组表达(rhFSH)和人尿提取(uhFSH)。由于糖基化结构差异，造成二者在半衰期、临床应用效果上差异显著。本项目利用多种糖基化结构分析和药物活性分析技术，首次研究rhFSH和uhFSH的糖基化差异及其生物活性相关性，揭示影响FSH活性的关键糖链。通过酶化学法对FSH糖基化结构进行改造，进一步深入研究关键糖链。并在糖工程学细胞中进行表达，获得活性更高且均一的FSH，以提高该药物质量，促进新型制剂的研发，为其他糖蛋白质药物的糖基化研究提供思路和策略。

卵泡刺激素(human follicle stimulating hormone，hFSH)，作为一种糖蛋白药物促进卵泡的分泌，在辅助生殖中发挥着关键作用。它由α、β两个亚基组成，每个亚基含有两个N-糖基化位点和带有唾液酸的复杂型N-糖链。作为药物，FSH主要有两种来源：CHO细胞重组表达的人卵泡刺激素(rhFSH)和尿提取的人卵泡刺激素(uhFSH)。大量的文献报道表明FSH糖基化显著影响其生物活性。α亚基的糖链对亚基间的整合、完整性、分泌和信号转导至关重要，而β亚基的糖基化则影响亚基间的整合和分泌。为了研究唾液酸对人卵泡刺激素(hFSH)生物活性的影响，本试验采用基于人卵巢颗粒样肿瘤细胞(human granulosa-like tumor cell line，KGN)的体外生物活性测定法和雌幼大鼠卵巢增重的体内生物活性测定法，测定经唾液酸酶酶切处理前后的重组人卵泡刺激素(recombinant human follicle stimulating hormone，rhFSH)和高纯度尿提取人卵泡刺激素(highly purified follicle stimulating hormone extracted from human urine，uhFSH)的体外、体内生物活性。结果表明，经唾液酸酶酶切处理后的rhFSH和uhFSH体内生物活性均丧失，并且唾液酸含量与hFSH体外生物活性呈负相关。通过进一步研究唾液酸酶酶切前后hFSH刺激与转染卵泡刺激素受体的中国仓鼠卵巢细胞（FSHR-CHO）环磷酸腺苷（cAMP）生成量的关系，观察到唾液酸含量越低，cAMP生成量越高，为研究hFSH唾液酸含量对其生物活性的影响提供参考。