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

项目 4：促性腺激素糖基化和功能的年龄相关变化

• 批准号: 10627095
• 负责人: GEORGE R BOUSFIELD
• 金额: $ 32.34万
• 依托单位: WICHITA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10627095
• 关键词: Adaptor Signaling Protein; Affect; Affinity; Age; Aging; Antibodies; Binding; Binding Sites; Biological; Biological Assay; Blood; Carbohydrates; Cell membrane; Collaborations; Complex; Cryoelectron Microscopy; Cultured Cells; Data; Enzyme-Linked Immunosorbent Assay; Estrogens; Exhibits; Family; Family Planning; Feedback; Fertility; Follicle Stimulating Hormone; Follicle Stimulating Hormone Receptor; G-Protein-Coupled Receptors; Gene Expression; Gene Expression Profile; Genes; Genitourinary system; Glycoproteins; Goals; Gonadotropins; Hormonal; Hormones; Human; Immunoassay; Impairment; In Vitro; Incubated; Individual; Knockout Mice; Knowledge; Ligand Binding; Ligand Binding Domain; Luteinizing Hormone; Measures; Mediating; Medicine; Menstrual cycle; Molecular Conformation; Morbidity - disease rate; Obesity; Oligosaccharides; Outcome; Ovarian; Ovarian Follicle; Ovary; Pathway interactions; Pattern; Perimenopause; Pharmaceutical Preparations; Physiological; Pituitary Gland; Polysaccharides; Population; Positioning Attribute; Primates; Resistance; Rhodopsin; Role; Rotation; Serum; Signal Transduction; Site; Steroids; Structure; Thyrotropin Receptor; Time; Transgenic Organisms; Transmembrane Domain; Urine; Uterine hemorrhage; Variant; Woman; access restrictions; age related; bone loss; bone mass; college; differential expression; drug development; extracellular; glycosylation; granulosa cell; hypothalamic pituitary gonadal axis; in vitro activity; in vivo; infertility treatment; inhibin; member; mullerian-inhibiting hormone; novel; older women; programs; psychologic; receptor; receptor binding; receptor function; receptor-mediated signaling; recruit; reproductive; reproductive senescence; response; tool; translational potential; vasomotor symptoms; young woman

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.

项目摘要/摘要 – 项目 4（布斯菲尔德） 我们的长期目标是了解碳水化合物如何调节卵泡刺激的生物活性 激素（FSH）。随着年龄的增长和循环 FSH 的增加，人类卵巢对 FSH 刺激产生抵抗力 浓度上升，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 从主要低糖基化的 hFSH21 和 hFSH18 发生变化（由于 分别在 Asn24 或 Asn7 处丢失 FSHβ 亚基 N-聚糖），形成主要完全糖基化的 hFSH24， 它拥有全部 4 个 N-聚糖。低糖基化 hFSH18 和 hFSH21 表现出更大的表观亲和力 FSHR，可以​​占据更多的 FSHR 位点，并且比完全糖基化的 hFSH24 更快地与 FSHR 结合。 我们的合作者已经证明 hFSH18 和 hFSH21 在体外表现出更大的生物活性，拯救 转基因 Fshb 缺失小鼠的生育力，并在颗粒细胞中表现出不同的基因表达模式。这 该提案的总体假设是，面对衰老的卵巢，从低功能状态的额外转变 糖基化 FSH18 和 FSH21 转变为完全糖基化 FSH24 进一步损害生育能力并有助于骨骼生长 损失。本提案的目的是通过 FSHR 结合研究这些差异的机制 具体目标如下： 1：开发多物种抗体的 FSH 糖型免疫分析。我们的 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存在差异。 hFSH18 和 hFSH21 诱导独特的基因表达谱这一事实使得开发 针对所有三种生理相关糖型 hFSH18、hFSH21 和 hFSH24 的特异性免疫测定。 2： 确定 FSHR/FSH-糖型结构。我们的工作假设是 FSH18、FSH21 和 FSH24 诱导不同的 FSHR 构象，从而产生有偏差的信号。该项目将净化FSHR和FSH/FSHR 复合物。低温电子显微镜（cryo-EM）将与赵王博士合作进行 贝勒医学院。高度纯化的 FSH 糖型、hFSH18、hFSH21 和 hFSH24 可用于 这两个目标。糖型特异性检测有望提供有关 FSH 糖型的新信息 在正常人类月经周期和女性年龄增长过程中会发生变化。 FSH 糖型/FSHR 结构是 有望揭示 FSH N-聚糖在充分激活 FSHR 中的作用。 该项目的潜在转化成果是用于测量 FSH 糖型的独特免疫测定法 FSHR 功能的知识，以促进增强或抑制 FSHR 功能的药物的开发。