FcRn Binds and Transports Albumin

FcRn 结合并运输白蛋白

• 批准号: 6926551
• 负责人: CLARK L ANDERSON
• 金额: $ 31.77万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6926551
• 关键词: affinity chromatography; albumins; antibody receptor; binding sites; calorimetry; chemical kinetics; clinical research; human tissue; immunofluorescence technique; laboratory mouse; pharmacokinetics; placental transfer; protein degradation; protein transport; radiotracer; receptor binding; surface plasmon resonance; tissue /cell culture

DESCRIPTION (provided by applicant): We tested a forgotten 40-year old hypothesis that the Brambell receptor, the MHC-related Fc receptor for IgG (FcRn), protects albumin from intracellular catabolic degradation just is it does IgG, prolonging the half-lives of both molecules. Our preliminary studies affirmed three predictions of this hypothesis, showing that albumin binds FcRn at low pH, just as does IgG, at a site distinct from the IgG binding site, forming a trimolecular complex. Preliminary experiments indicate direct binding of albumin to the native receptor; SPR experiments show KD in fM range. Further, we found that FcRn-deficient (KO) mice degrade albumin more rapidly than WT mice, and that the serum albumin concentration in FcRn-deficient mice is about half that of WT controls. These data infer that many fold more albumin molecules are protected from degradation per IgG molecule per unit time, and that FcRn may somehow be involved in albumin biosynthesis. Our specific aims are two fold. First, we will characterize the molecular interaction of albumin with FcRn by determining affinity, kinetic rate constants, specificity, and stoichiometry of interaction. Second, we will test six predictions or extensions of the hypothesis; namely, that native FcRn from tissue will bind albumin, that albumin may be transported from mother to offspring, that FcRn mediates trans-endothelial flux of albumin, that FcRn participates in hepatocyte production of albumin, that albumin transport across cell in vitro is FcRn mediated, and that a family deficient in albumin and IgG because of accelerated degradation rates of both molecules have a faulty FcRn gene. We apply a broad range of methods encompassing many disciplines. In the first aim, mostly biochemical, we use surface plasmon resonance, radioligand binding assays, size exclusion chromatography, isothermal titration calorimetry, and affinity chromatography. The second aim, which includes several physiological goals, employs pharmacokinetic analysis, in vivo transport studies in KO mice, tissue culture, immunofluorescence, and molecular biology. The implications of these studies extend far on a broad range of health and disease topics. These include the homeostatic regulation of serum albumin concentration; the flux of albumin across endothelium between plasma and extravascular compartments arid across other tissue barriers; the mechanism of hepatocyte albumin production; the placental transport of proteins; and the evolution of the MHC family of proteins.

描述（由申请人提供）：我们测试了一个被遗忘了40年的假设，即Brambell受体（IgG的MHC相关Fc受体（FcRn））保护白蛋白免受细胞内分解代谢降解，就像它保护IgG一样，延长了两种分子的半衰期。我们的初步研究证实了这一假设的三个预测，表明白蛋白在低pH下结合FcRn，就像IgG一样，在不同于IgG结合位点的位点结合，形成三分子复合物。初步实验表明白蛋白与天然受体的直接结合; SPR实验显示KD在fM范围内。此外，我们发现FcRn缺陷（KO）小鼠比WT小鼠更快地降解白蛋白，并且FcRn缺陷小鼠的血清白蛋白浓度约为WT对照的一半。这些数据推断，单位时间内每个IgG分子保护多许多倍的白蛋白分子免于降解，并且FcRn可能以某种方式参与白蛋白生物合成。 我们的具体目标有两个方面。首先，我们将通过测定亲和力、动力学速率常数、特异性和相互作用的化学计量来表征白蛋白与FcRn的分子相互作用。其次，我们将测试六个预测或假设的扩展;即，来自组织的天然FcRn将结合白蛋白，白蛋白可以从母体转运到后代，FcRn介导白蛋白的跨内皮流动，FcRn参与白蛋白的肝细胞产生，体外白蛋白跨细胞转运是FcRn介导的，并且由于白蛋白和IgG两种分子的加速降解速率而缺乏这两种分子的家族具有缺陷的FcRn基因。 我们采用涵盖许多学科的广泛方法。在第一个目标，主要是生物化学，我们使用表面等离子体共振，放射性配体结合试验，尺寸排阻色谱，等温滴定量热法，亲和色谱。第二个目标包括几个生理目标，采用药代动力学分析、KO小鼠体内转运研究、组织培养、免疫荧光和分子生物学。 这些研究的影响范围很广，涉及健康和疾病问题。这些包括血清白蛋白浓度的稳态调节;白蛋白穿过血浆和血管外隔室之间的内皮和穿过其他组织屏障的通量;肝细胞白蛋白产生的机制;蛋白质的胎盘转运;以及MHC家族蛋白质的进化。