臺灣博碩士論文加值系統

SCUBE1屬於一個嶄新的SCUBE醣蛋白家族，具有分泌且可附著於細胞膜上的特性， SCUBE1基因轉譯出約含1000個胺基酸之蛋白質，其蛋白質結構包含: N端之訊號序列 (signal peptide)、9個似表皮生長因子之重複片段 (EGF-like repeats)、1個間隔區段 (spacer region)、3個富含半胱胺酸之模組 (cysteine-rich motif) 和C端含有CUB之區域 (CUB domain)。我們先前的研究利用斑馬魚動物模式探討scube1基因在胚胎發育中的功能，在細胞膜上的SCUBE1與BMP受體形成複合體，透過調控BMP活性而影響胚胎時期的血球生成。然而，目前SCUBE1附著於細胞膜的機制仍未知，且其N-醣基化的功能尚未被驗證。此外，在小鼠腎臟損傷模式中，腎小管週邊微血管內皮細胞會增加SCUBE1的表達，並以旁分泌的方式 (paracrine) 促使受損之腎臟修復。但是，腎臟上皮細胞的SCUBE1於腎臟損傷和修復之重要性及其確切的保護機制為何，仍待進一步釐清。本論文分為兩個部分: 第二章詳細探討SCUBE1附著於細胞膜的機制；第三章利用Scube1-Δ2老鼠驗證SCUBE1於腎臟病理學之角色。
在第二章中，我們提供了嶄新的機制解釋SCUBE1如何黏附於細胞膜上，並證實SCUBE1之N-醣基化修飾在斑馬魚早期的造血系統具有重要生物功能。本章研究利用分子生物實驗技術，找出間隔區段內一段帶有大量正電荷的區間 (胺基酸501-550) 可藉由靜電吸引力與帶負電荷之硫酸乙醯肝素蛋白聚醣結合而黏附於細胞膜上；此外，以肽-N端切醣素 (PNGaseF) 去除富含半胱胺酸模組內之N-醣化修飾，可得知其N-醣化修飾對於黏附於細胞膜上之重要性，且可能是透過與凝集素 (lectin) 結合而黏附在細胞膜上；而SCUBE1必須黏附於膜上才能調節BMP訊號傳遞。最後，利用斑馬魚模式驗證SCUBE1之N-醣化修飾在活體的重要性，利用antisense morpholino (MO) 的方式抑制scube1的轉譯，減少scube1表達會進一步抑制胚胎造血和紅血球標的基因的表達 (scl and gata1)，若同時注射野生型或N-醣化缺失之scube1訊息核糖核酸 (mRNA) 於缺乏scube1表現的斑馬魚胚胎內，僅野生型而非N-醣化缺失之scube1訊息核糖核酸能恢復造血和紅血球標的基因表達。我們的研究說明了SCUBE1至少藉由兩種不同的機制附著於細胞膜上，也利用斑馬魚模式闡明了SCUBE1之N-醣基化修飾於活體的重要性。
在第三章中，我們驗證了SCUBE1能保護腎臟免於急性腎臟損傷。在本章中，我們將WT和Scube1-Δ2老鼠施予典型的腎臟缺血再灌流手術，分析腎臟病理組織學、腎臟損傷生物指標、BMP訊號傳遞、細胞凋亡、細胞修復，與WT老鼠比較，Scube1-Δ2老鼠具有較嚴重的病理特徵。藉由分子生物和生物化學的分析發現，SCUBE1能與BMP7及其受體形成複合體，並藉此調控BMP訊息傳遞。我們的研究首度證實了腎臟損傷會增加SCUBE1表達，進而增強BMP7的訊息傳遞而達到保護作用。
本論文提供了SCUBE1附著於細胞膜上的機制，並驗證了其N-醣基化修飾的重要性，同時利用腎臟缺血再灌流模式和Scube1-Δ2老鼠，驗證SCUBE1在腎臟損傷過程中藉由增強BMP訊息傳遞而具有保護的作用。

SCUBE1 (signal peptide-CUB [complement protein C1r/C1s, Uegf, and Bmp1]-EGF [epidermal growth factor] domain-containing protein 1) is the founding member of a secreted and membrane-associated SCUBE protein family. This gene codes for protein of approximately 1,000 amino acids forming at least 5 domains: an NH2-terminal signal peptide sequence, 9 tandem repeats of EGF-like motifs, a large spacer region followed by 3 N-glycosylated cysteine-rich (CR) repeats and one CUB domain at the COOH terminus. Our recent zebrafish genetic study revealed that scube1 is critical for and function at the top of regulatory hierarchy of primitive hematopoiesis by acting as a bone morphogenetic protein (BMP) co-receptor on cell-surface to augment BMP signaling. However, the membrane-anchoring mechanism of SCUBE1 and functional implication of its N-linked glycosylation remain largely unknown. In addition, although our previous study provided evidence that the SCUBE1 protein may constitute an critical regulatory protein operant within the renal vascular network exerting reparative effects on adjacent renal tubules in a paracrine fashion, the importance of the renal epithelial SCUBE1 protein in adult kidney injury and endogenous repair remains elusive. This dissertation is composed of two parts: Chapter 2 is focus on dissecting the membrane-tethering mechanisms of SCUBE1; in Chapter 3, we generated new genetic deletion Scube1-Δ2 mutant mice to further elucidate the renal pathophysiological roles of SCUBE1.
In Chapter 2, we describe novel mechanisms in targeting SCUBE1 to the plasma membrane and demonstrate that N-glycans are required for SCUBE1 functions during primitive hematopoiesis in zebrafish. Molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide-N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. In addition, the surface-tethering at the plasma membrane of SCUBE1 is critical for its co-receptor function in promoting BMP signaling. Lastly, injection of mRNA encoding zebrafish wild-type but not N-glycan–deficient scube1 restores the expression of hematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. Together, our results revealed SCUBE1 operating by two distinct membrane-anchoring mechanisms through its spacer region and the CR domain, and genetic rescue experiments suggested that N-glycosylation plays an important role in SCUBE1 function in vivo.
In Chapter 3, we identified the protective role of SCUBE1 in acute kidney injury. A new genetic Scube1-Δ2 mutant mouse strain was generated and subjected to renal ischemia-reperfusion injury as compared to WT controls. We found that Scube1-Δ2 mutant mice had a severe renal histopathologies, elevated biomarker levels for acute renal injury, and reduced BMP signaling in association with decreased proliferation and increased apoptosis in renal tubular cells compared with wild-type mice. Molecular and biochemical analyses revealed that SCUBE1 could interact with BMP7 and its receptors, and may act as a BMP co-receptor to augment BMP7 signaling. Together, our data demonstrate for the first time that the protective BMP7 signaling is enhanced by the stress-inducible SCUBE1 after renal I/R and suggest potential targeted approaches for acute kidney injury.
In summary, our studies identified the membrane-tethering mechanisms and in vivo biological relevance of N-glycans of SCUBE1 protein. We also revealed that SCUBE1 has the protective effect in acute kidney injury via modulating BMP signaling.

Table of Contents...…………………………………………………………………….i
List of Figures and Tables…………………………………….……………………....iii
Chinese Abstcact…………………………………………………………...………....vi
English Abstract……………………………………………...………………...……viii
Chapter 1. Introduction
1. SCUBE gene family………………….…………………………………………2
2. Rationale for dissecting the membrane-anchoring mechanism(s) of SCUBE1 and the biological function of its N-linked glycosylation……………………………3
3. Acute kidney injury (AKI).…………………………………………………..…4
4. BMP7 signaling pathway in renal disease………………………………………5
5. Rationale for investigating the role of SCUBE1 in AKI…………………..……7
Chapter 2. Electrostatics and N-Glycan-mediated membrane tethering of SCUBE1 is critical for promoting bone morphogenetic protein signaling
1. Summary………………………………………………………………………..9
2. Introduction and specific aims…………………………...……………………10
3. Materials and methods………………………………………………………...12
4. Results………………………………………………………………………... 16
5. Discussion……………………………………………………………………..24
Chapter 3. SCUBE1 protects against acute kidney injury by promoting BMP signaling
1. Summary………………………………………………………………………28
2. Introduction and specific aims………………………………………………...29
3. Materials and methods………………………………………………………... 31
4. Results………………………………………………………………………... 36
5. Discussion……………………………………………………………………..42
Chapter 4. Future Perspectives……………………………………………………..45
References…………………………………………………………….…………….. 48

FIGURE 1. Membrane association of human SCUBE1 is mediated by the spacer region and cysteine-rich (CR) motifs……………………………………………59
FIGURE 2. Cell surface binding assay of SCUBE1 domain-containing GST fusion proteins………………………………………………………………..…60
FIGURE 3. The spacer region and CR domain are involved in the cell surface binding of SCUBE1……………………………………….…………………...…61
FIGURE 4. Localization of SCUBE1 within the caveolin-1-enriched raft microdomain and interaction of SCUBE1 with caveolin-1……….…………………… 63
FIGURE 5. The membrane-tethering ability of SCUBE1 is critical for modulating BMP signaling……………………………………………………………….…65
FIGURE 6. Amino acids 501-550 within the spacer region are critical for membrane-binding ability………………………………………………………....…66
FIGURE 7. A segment of positively charged amino acids in the spacer region is necessary for its tethering on the cell surface …………………….………67
FIGURE 8. The spacer region of SCUBE1 directly binds heparin…………………..69
FIGURE 9. N-glycosylation of recombinant CR protein fragment confers its membrane binding ability …………………………….………………………….… 70
FIGURE 10. N-linked oligosaccharides of the CR motif are crucial for its membrane association Localization of SCUBE1 within the caveolin-1-enriched raft microdomain and interaction of SCUBE1 with caveolin-1………………71
FIGURE 11. N-glycosylation is required for zebrafish Scube1 function in vivo…….72
FIGURE 12. Scube1-WT not Scube1-ΔN can rescue scube1 morphants……………74
FIGURE 13. Lectin blotting profile of SUBE1………………………………………75
FIGURE 14. The spacer region of SCUBE2 and SCUBE3 directly binds heparin……76
FIGURE 15. SCUBE1 mRNA and protein levels were upregulated after renal ischemia-reperfusion (I/R) injury……………………………………………..……77
FIGURE 16. Generation of a targeted deletion (Δ2) allele of Scube1 gene………….78
FIGURE 17. Targeted deletion of Scube1 was confirmed at both mRNA and protein levels……………….…………………………………………………….79
FIGURE 18. Deletion Δ2 mice showed severe histological features after renal I/R injury……………….……………………………………………………80
FIGURE 19. Increased polymorphonuclear leukocyte infiltration and elevated proinflmmatory mediator tumor necrosis factor α (TNF-α) expression in Δ2 and wild-type (WT) control mice…………………..……………….……81
FIGURE 20. Expression of acute renal injury biomarkers is higher in Δ2 than WT mice………………………………………………………………….…..82
FIGURE 21. SCUBE1 binds BMP7 ligand / receptors and augments BMP signaling…………………………………………………………………83
FIGURE 22. Reduced BMP7 signaling in the Δ2 mouse embryonic fibroblasts (MEFs) or Δ2 kidneys…………………………………………………………….85
FIGURE 23. Effect of renal I/R on the expression of BMP7 and BMPRs in WT and Scube1-Δ2 mice………………………………………………………..86
FIGURE 24. Δ2 mice showed more renal tubular cell apoptosis after I/R injury………………………………………………………………….…87
FIGURE 25. Δ2 mice showed decreased renal tubular cell proliferation after I/R injury…………………………………………………………………….88
FIGURE 26. SCUBE1 overexpression enhances BMP signaling and reduces hypoxia/reoxygenation-induced apoptosis in Δ2 proximal tubular epithelial cells (PTECs)……………………………………….……………………89
FIGURE 27. In vitro hypoxia-reoxygenation (H/R) induced Scube1 mRNA and protein expression in HK-2 cells…………………………………………..……..90
FIGURE 28. SCUBE1 mRNA and protein levels were upregulated after chronic unilateral ureteral obstruction (UUO) injury……………………………..91
TABLE 1. Primers used for RT-PCR, Q-PCR and genotyping analyses………………92
TABLE 2. Gender and genotype distribution of Scube1+/Δ2 intercross……………..93

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