Project 4: Age-Related Changes in Gonadotropin Glycosylation and Function

项目概要/摘要-项目4（Bousfield） 我们的长期目标是了解碳水化合物如何调节卵泡刺激素的生物活性， 激素（FSH）。随着年龄的增长和循环FSH的增加，人类卵巢对FSH刺激产生抗性。 雌激素浓度升高，使血清雌激素浓度保持正常，直到最后一次月经周期前2年。 大约在同一时间，垂体FSH从主要是低糖基化的hFSH 21和hFSH 18（由于 分别在Asn 24或Asn 7处FSHβ亚基N-聚糖的损失）转化为主要完全糖基化的hFSH 24， 其具有全部4个N-聚糖。低糖基化的hFSH 18和hFSH 21表现出更大的表观亲和力， FSHR可以占据更多FSHR位点，并比完全糖基化的hFSH 24更快地与FSHR结合。 我们的合作者已经证明hFSH 18和hFSH 21在体外表现出更大的生物活性，拯救 在转基因Fshb基因敲除小鼠中的生育力，并在颗粒细胞中表现出不同的基因表达模式。的 这一建议的总体假设是，面对衰老的卵巢，从低- 糖基化的FSH 18和FSH 21转化为完全糖基化的FSH 24进一步损害生育力， 损失本提案的目的是研究FSHR结合中这些差异的机制， 具体目标如下：1.开发多种类抗体的FSH糖型免疫测定法。我们 工作假设是完全糖基化和部分糖基化的FSH糖型分泌模式在女性中不同。 hFSH 18和hFSH 21诱导独特的基因表达谱的事实使得开发 所有三种生理学相关糖型hFSH 18、hFSH 21和hFSH 24的特异性免疫测定。第二章： 确定FSHR/FSH-糖型结构。我们的工作假设是，FSH 18，FSH 21和FSH 24 诱导导致偏置信号不同FSHR构象。本项目将对FSHR和FSH/FSHR进行纯化 配合物低温电子显微镜（cryo-EM）将与Zhao Wang博士合作进行， 贝勒医学院。高度纯化的FSH糖型hFSH 18、hFSH 21和hFSH 24可用于 两个目标。糖型特异性检测有望提供有关FSH糖型的新信息 在正常的人类月经周期和女性年龄的变化。FSH糖型/FSHR结构是 预期揭示FSH N-聚糖在完全激活FSHR中的作用。 该项目的潜在转化结果是测量FSH糖型的独特免疫测定法 和FSHR功能的知识，以促进增强或抑制FSHR功能的药物的开发。

Project Summary/Abstract – Project 4(Bousfield) Our long-term goal is to understand how carbohydrate modulates the biological activity of follicle-stimulating hormone (FSH). The human ovary becomes resistant to FSH stimulation with age and circulating FSH concentrations rise, keeping serum estrogen concentrations normal until 2 years before the final menstrual cycle. Around the same time pituitary FSH changes from predominantly hypo-glycosylated hFSH21 and hFSH18 (due to loss of FSHβ subunit N-glycan at Asn24 or Asn7, respectively) to a predominantly fully-glycosylated hFSH24, which possesses all 4 N-glycans. Hypo-glycosylated hFSH18 and hFSH21 exhibit greater apparent affinity for the FSHR, can occupy more FSHR sites, and associate with FSHR more quickly than fully-glycosylated hFSH24. Our collaborators have demonstrated hFSH18 and hFSH21 exhibit greater biological activity in vitro, rescue fertility in transgenic Fshb-null mice, and exhibit distinct patterns of gene expression in granulosa cells. The overall hypothesis of this proposal is that in the face of a senescing ovary the additional switch from hypo- glycosylated FSH18 and FSH21 to fully-glycosylated FSH24 further compromises fertility and contributes to bone loss. The objective of this proposal is to study the mechanisms for these differences in FSHR binding via the following specific aims: 1: Develop FSH glycoform immunoassays with multi-species antibodies. Our working hypothesis is that fully- and partially glycosylated FSH glycoforms secretion patterns diverge in women. The fact that hFSH18 and hFSH21 induce unique gene expression profiles makes it imperative to develop specific immunoassays for all three physiologically relevant glycoforms, hFSH18, hFSH21, and hFSH24. 2: Determine FSHR/FSH-glycoform structures. Our working hypothesis is that FSH18, FSH21, and FSH24 induce different FSHR conformations that result in biased signals. The Project will purify FSHR and FSH/FSHR complexes. Cryogenic electron microscopy (cryo-EM) will be conducted in collaboration with Dr. Zhao Wang at Baylor College of Medicine. Highly purified FSH glycoforms, hFSH18, hFSH21, and hFSH24 are available for both aims. Glycoform-specific assays are expected to provide novel information regarding FSH glycoform changes during the normal human menstrual cycle and as women age. FSH glycoform/FSHR structures are expected to reveal the role of FSH N-glycans in fully activating the FSHR. The potential translational outcomes of this project are unique immunoassays for measuring FSH glycoforms and knowledge of FSHR function to facilitate development of drugs to enhance or inhibit FSHR function.