參與Wnt訊息傳遞之Dickkpof-2 (DKK2) 蛋白質的轉譯後修飾與運送

分泌性蛋白質Wnt：存在細胞之間特定的訊息傳遞途徑，扮演關鍵性的中介傳遞者。Wnt基因家族的成員在物種之間具有演化高度保留的特性；破壞或剔除Wnt訊息傳遞途徑會導致發育畸形、器官退化以及癌症的發生，這些生物現象顯示Wnt訊息傳遞途徑的重要性。Wnt所影響的層面廣泛且重要，主要包含：胚胎發育、成體的組織維持等。另外，Wnt的活性會受到一系列分泌性蛋白質的影響，其中包含也具有演化高度保留性質的Dickkopf (DKK)蛋白質家族，而根據目前的研究發現，DKK家族成員也參與調控胚胎發育以及影響癌症形成。　　本實驗室先前的痛風臨床分析，鑑定出座落於DKK2基因的單一核苷酸多型性(Single Nucleotide Polymorphism)：DKK2 exon_01_+791 G>T。這個單一核苷酸多型性的變異會導致DKK2蛋白質的第二十九個胺基酸從甘胺酸(Glycine)取代為纈胺酸(Valine)，突變所取代的胺基酸位於DKK2訊息胜肽的端點，由進一步生物資訊軟體SignalP分析的結果顯示：含有SNP而取代為纈胺酸的DKK2蛋白質訊息胜肽被辨識切割的能力會降低。分泌性DKK2蛋白質，是訊息傳遞的配體(Signaling ligand)，藉由分泌到細胞外而調控Wnt典型訊息傳遞(beta-catenin signaling)的活性。因此，基於上述的預測以及DKK2訊息調控功能性，我們假設這個SNP引起的胺基酸變異會影響DKK2的分泌功能，使得DKK2訊息傳遞途徑失調，最後可能導致疾病的發生。為了研究這個SNP潛在的影響功能，我們利用Flp-In T-REx System建立可誘發表現的DKK2質體所形成的穩定細胞株(質體包括：pcDNA5/FRT/TO DKK2::FlagC的野生型(G)、突變型(T)以及僅有載體的控制組pcDNA5/FRT/TO FlagC)。質體經由專一性重組作用嵌入基因體，再進行抗藥性篩選，最後用南方雜合反應來驗證穩定細胞株的表現套數以及是否正確嵌入基因體中FRT 序列位置。分析不同SNP的穩定細胞株DKK2蛋白質在細胞質和細胞培養基表現量的差異；另一方面，透過典型Wnt3a訊息下游的專一性啟動子(TCF promoter)作報導基因的分析，來定量野生型與突變型DKK2抑制訊息傳導的強度是否有差異。西方墨點法的結果顯示DKK2在細胞質與培養基中存在多種分子量的型態，根據目前DKK2的研究，這些不同分子量的型態可能是由蛋白質切割(proteolytic processing)以及轉譯後修飾(post-translation modification)作用共同造成；我們還發現野生型在培養基內大分子量(34　kD)的表現量較多，而較小分子量(13.5 kD)的表現量較少(相對於突變型)；在24小時誘發後，突變型在細胞質內的33.5 kD型態表現量較多，而較小分子量30.5 kD型態表現量較少。　　為了釐清多型態分子量的意涵，使用胺基端去醣基酶(PNGase F)驗證DKK2的醣化修飾；透過位點突變第52個胺基酸來確認這個高保留性的醣化修飾位置；刪除DKK2訊息胜肽(Signal Peptide)推導蛋白質運送以及修飾的先後關係。實驗結果顯示DKK2訊息胜肽(signal peptide)的改變會造成DKK2蛋白質分泌過程中間產物表現量的差異。因此，我們提出解釋模型：SNP可能會影響DKK2修飾與分泌的速率，而這現象或許會影響DKK2的功能進而影響Wnt訊息傳遞。

關鍵字

轉譯後修飾；醣化； DKK2

並列摘要

Members of the highly conserved Wnt secreted protein family encode critical mediators of cell-cell signaling events. Wnt gene family influence many aspects of embryonic development, and are required for adult tissue maintenance. Previous studies have also indicated that perturbation of the Wnt signaling is associated with human degenerative diseases and cancers. Wnt activity is regulated by several secreted proteins, including the conserved Dickkopf (DKK) protein family. DKK-related family members also play a role in embryonic development and cancers. In previous clinical studies, we have identified a SNP in DKK2 exon_01_+791 G>T. This nucleotide change leads to Gly to Val substitution at amino acid position 29. DKK2 is a secretory glycoprotein which modulates canonical Wnt signaling levels (beta-catenin). Therefore, we hypothesized this amino acid change could influence the secretion of DKK2. In order to explore this SNP function, we established in vitro system of Flp-In T-REx-293 cell line containing inducible DKK2 expression plasmids (pcDNA5/TO/FRT-DKK2-flag: wild type, mutant type, and vector-only).These plasmids were generated by specific Flp-mediated recombination at a specific genome site. The copy number was checked by Southern blot. Then, we compared DKK2 protein level in cell lysate and medium to demonstrate the influence of the SNP. On the other hand, canonical Wnt3a signaling (TCF/ beta-catenin) reporter assay was performed to demonstrate the difference between wild type and mutant type of DKK2-signaling. Immunoblots revealed multiple forms of DKK2 in cell lysate and medium. In medium the amount of large-size DKK2(34 kD) seemed higher in wild type but small-size of DKK2(13.5 kD) seemed more in mutant type.On the other hand, in cell the amount of large-size form (33.5 kD) was higher in mutant type and small-size form (30.5 kD) was more in wild type after 24hr induction. According to previous studies, this size variation may be a result of post-translation modification or proteolytic processing. In fact, we discovered that these size variations are due to glycosylation by glycodidase mobility shift assay. In conclusion, alteration in the signal peptide of DKK2 affects the different amount of intermediates in the DKK2 protein sorting pathway. We deduce a hypothisis that the SNP will cause different rate of DKK2 modification or secretion. These post-translational events may affect the function of DKK2 and, therefore, the Wnt-signaling.

並列關鍵字

DKK2 ； Dickkpof ； Wnt ； Glycosylation ； N-linked glyconsylation

參考文獻

Aguilera, O., Munoz, A., Esteller, M., and Fraga, M. F. (2007). Epigenetic alterations of the Wnt/beta-catenin pathway in human disease. Endocr Metab Immune Disord Drug Targets 7, 13-21.

Brott, B. K., and Sokol, S. Y. (2002). Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins. Mol Cell Biol 22, 6100-6110.

Cheng, L. S., Chiang, S. L., Tu, H. P., Chang, S. J., Wang, T. N., Ko, A. M., Chakraborty, R., and Ko, Y. C. (2004). Genomewide scan for gout in taiwanese aborigines reveals linkage to chromosome 4q25. Am J Hum Genet 75, 498-503.

Chou, C. T., and Lai, J. S. (1998). The epidemiology of hyperuricaemia and gout in Taiwan aborigines. Br J Rheumatol 37, 258-262.

Emanuelsson, O., Brunak, S., von Heijne, G., and Nielsen, H. (2007). Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2, 953-971.

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參與Wnt訊息傳遞之Dickkpof-2 (DKK2) 蛋白質的轉譯後修飾與運送

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