Prolyl-3 Hydroxylation of Type IV Collagen in Kidney Development

描述（由申请人提供）：脯氨酰-3羟化酶是最近鉴定的参与胶原蛋白翻译后修饰的酶家族。原纤维胶原具有经历脯氨酰-3羟基化的单个残基，而IV型基底膜胶原具有12-15个修饰的残基。肾脏由基底膜组成，具有独特的胶原异源三聚体，有助于其特定功能，包括肾小球基底膜、系膜基质和肾小管基底膜。初步的原位杂交数据显示脯氨酰-3羟化酶2和3（P3 H2和P3 H3）在肾脏内以不同的分布表达。P3 H2特异于肾小球，而P3 H3定位于中央系膜分布、小管和集合系统。我们建议通过产生针对每个基因的靶向敲除小鼠来研究P3 H2和P3 H3在肾脏发育中的生物学功能。靶向策略将是通过同源重组删除内部外显子，导致开放阅读框的破坏。这些小鼠的研究将包括组织学、电子显微镜、尿蛋白和血清肌酐测量以及用于评价基因表达的冷冻组织收集。将在多个出生后和胚胎时间点研究肾脏表型。在遗传性肾炎患者的基因中已经鉴定出新的P3 H3序列变异。这些序列变体定位于功能性双加氧酶结构域，并且在整个脊椎动物中是保守的。我们计划研究这些变体的功能相比，野生型重组人蛋白在昆虫细胞中产生的。体内试验将使用重组人胶原蛋白作为底物，测量与NADH氧化偶联的琥珀酸产生。如果这些变异是有害的，我们将继续分析更大的慢性肾脏疾病人群，以评估异常脯氨酰-3羟基化在该组中发挥的作用。相关性：在肾脏中，细胞周围膜中的专门胶原蛋白有助于肾脏在体内血液和尿液空间之间过滤的功能。这项研究的重点是脯氨酰-3羟化酶，蛋白质，使胶原蛋白的变化，影响其形式和功能。了解这些蛋白质在肾脏中的作用可能会导致改善筛查方法，早期检测和慢性肾脏疾病患者的新治疗方法。

DESCRIPTION (provided by applicant): Prolyl-3 hydroxylases are a recently identified family of enzymes involved in post-translational modification of collagens. Fibrillar collagens have a single residue that undergoes prolyl-3 hydroxylation, whereas type IV basement membrane collagen has 12-15 modified residues. The kidney is composed of basement membranes with unique collagen hetero-trimers contributing to their specific functions, including the glomerular basement membrane, mesangial matrix, and tubular basement membranes. Preliminary in- situ hybridization data shows prolyl-3 hydroxylases 2 & 3 (P3H2 and P3H3) are expressed in distinct distributions within the kidney. P3H2 is specific to the glomeruli, while P3H3 localizes to a central mesangial distribution, tubules, and collecting system. We propose to study the biologic function of P3H2 and P3H3 in kidney development by generating targeted knock-out mice for each gene. The targeting strategy will be deletion of interior exons via homologous recombination, leading to disruption of the open reading frame. Study of these mice will include histology, electron microscopy, urine protein and serum creatinine measurements, and frozen tissue collection for evaluation of gene expression. The kidney phenotype will be studied at multiple post-natal and embryonic time points. Novel P3H3 sequence variations have been identified in the genes of patients with hereditary nephritis. These sequence variants localize to the functional dioxygenase domain, and are conserved throughout vertebrates. We plan to study the function of these variants compared to wild-type recombinant human protein produced in insect cells. The in-vivo assay will use recombinant human collagen as a substrate, measuring succinate production coupled to NADH oxidation. If these variants are deleterious, we will proceed to analyze a larger population with chronic kidney disease to evaluate what role abnormal prolyl-3 hydroxylation plays in this group. Relevance: In the kidney, specialized collagens in the membranes surrounding cells aid in the kidney's function of filtering between the blood and urinary spaces in the body. This study focuses on prolyl-3hydroxylases, proteins that make changes to collagen that affects its form and function. Understanding the role of these proteins in the kidney may lead to improved screening methods, earlier detection, and new treatments for patients with chronic kidney disease.