| 摘要: | C型凝集素(CLEC)是一類被認為受鈣離子所調控的醣蛋白，對於抗原辨識、免疫調節啟動等扮演重要的角色。此類蛋白含有一段約130個氨基酸序列的醣辨識區，根據其蛋白構型，CLEC家族共區分成17類。而CLEC18被歸在第16類，其蛋白構型主要包含三個功能區：CAP/SCP/TAPS、EGF和CRD。人類的CLEC18座落在染色體16q22的位置，包含三個序列高度重複的基因：CLEC18A、CLEC18B和CLEC18C。
免疫螢光染色發現CLEC18除了分布在細胞內的高基氏體(Golgi apparatus)、內質網(endoplasmic reticulum)和初級胞內體(early endosome)，也會分泌至細胞外(extracellular milieu)。在CLEC18家族的CRD區段第339號和第421號位置發生氨基酸改變時，會影響聚糖(glycan)的親和專一性。最近的研究更指出，CLEC18第339號位置發生氨基酸改變，與TLR3仍能形成共同受體(co-receptor)且能強化免疫反應以抵抗病毒的感染。此外，過去曾報導CAP super family 具有攜帶並運送脂質之功能，然而在CLEC18上的CAP/SCP/TAPS 功能區是否具有類似的功能，以及其所在生物體中所扮演的角色以及其相關的性狀，目前仍未有一個系統性的研究。
本研究首先利用人類生物檢體資料庫的分析，進行CLEC18 基因組的全表型關聯性研究。接著，尋找具有潛在影響CLEC18基因表現量及蛋白質功能的基因多型性（single nucleotide polymorphism, SNP），進一步擴大樣本數與結合眾多不同資料庫，延伸至人類基因多型性所連結之多體學性狀(包含轉錄體、蛋白質體、代謝體等)與臨床結果，進行一系列的探討。此外，我們也進行老鼠實驗，觀察將Clec18a基因剔除後所引起之相關表型紀錄 ，並利用293T細胞，進行過度表現(over-expressed) CLEC18A，觀察對於脂質生成的影響。
在本研究中，我們首先發現了多個CLEC18A 基因多型性與血液中尿酸的表現量呈現顯著相關。接著我們找到一具有功能性影響的基因多型性，位於CLEC18A基因上的rs75776403，其轉錄成的第151號位置氨基酸(p.T151M)正座落於CAP/SCP/TAPS 功能區內。結果顯示，rs75776403 所造成的氨基酸改變，會造成CAP/SCP/TAPS功能的受損，影響了對於脂質(PA和PS)的親和力。rs75776403並與身高、心肌病變的風險以腎功能指數還有荷爾蒙如甲狀腺重要的基因(如TG、THRB等)等，都有相關。我們在老鼠中發現，Clec18a 失活的老鼠，約二到三週就會死亡，身體較為瘦小，流體/體重(free -fluid/body weight)比值也較高，且皮下觀察不見白脂肪（white adipocyte）的堆積。在細胞實驗中我們亦發現，細胞在starvation 的情況下，過度表現CLEC18A會促進脂肪油滴(lipid drops)的生成以及peroxisomes的增加。
本研究在CLEC18A與生理上扮演的角色相關性提供重要的線索，尤其對於脂質的結合與生成，以及透過荷爾蒙的改變可能造成生長發育代謝相關等影響。未來在研究上，仍須進一步進行驗證，深入了解CLEC18A如何促進脂質生成的機轉以及荷爾蒙激素的改變。
C-type lectins (CLEC) superfamily comprises 17 groups, which play prominent roles in pathogen recognition, immune regulation, and other physiological mechanisms. The presence of C-type lectin domain (CTLD) is the character of CLEC, which is considered as carbohydrate-recognition domain in Ca2+ dependent lectin. CLEC18 gene family belongs to the group XVI, which consists of a C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 or Sperm-coating protein or Tpx antigen 5/pathogenesis related-1/Sc7 (CAP/SCP/TAPS) domain, epidermal growth factor (EGF) and EGF-like domains, as well as carbohydrate recognition domain (CRD). In human, the CLEC18 gene family is located on chromosome 16q22, which contains 3 highly conserve genes (CLEC18A, CLEC18B, CLEC18C).
CLEC18 are localized in intracellular organelles such as Golgi apparatus, endoplasmic reticulum (ER), early endosomes, and can also be found in the extracellular milieu. The polymorphic residue of p.S339R and p.D421N in CRD domain of CLEC18 affected glycan-binding ability. Previous studies have linked the implication of CLEC18A and CLEC18A(S339R) in protecting against H5N1 by promoting host immune. In addition, CAP superfamily have been shown to have the lipid binding ability. The functions of CAP/SCP/TAPS domain of CLEC18 remain unknown and a systemic investigation of CLEC18 in pathophysiological roles remain lacking.
In this research, phenome-wide association study (PheWAS) was conducted to identify the phenotypic associations of CLEC18 genetic polymorphisms. Next, we identified a deleterious SNP in CLEC18 gene and further characterized the associated profiles and enriched pathways with multiple omics (including transcriptome, proteiome, metabolism, and phenome) by a varies of human datasets. The effects of Clec18a deficiency were observe in the mice model. In the cell-based experiments, CLEC18A were overexpressed in 293T cell line to evaluate its effects.
From PheWAS results of CLEC family genes revealed the significant correlation between CLEC18A genetic polymorphisms and uric acid levels in serum. Next, we identified a missense cis-acting expression quantitative trait locus in CLEC18A (rs75776403) in which the altered residue (p.T151M) disrupts the lipid-binding (PA and PS) ability of the CAP/SCP/TAPS domain. The altered allele carriage led to a metabolic and proliferative shift, as well as hormone changes, therefore determines human anthropometrics (body height), and kidney traits. In the mice model, early expiration and reduced body size were occurred when Clec18a deficiency. A lack of white adipocytes accumulation were observed in the Clec18a null mice. However, when overexpressed CLEC18A in the 293T cell line, we found that the lipid drops were increased, as well as peroxisomes.
This study boosts our understanding of CLEC18A potential pleiotropic roles especially in lipid binding and lipid accumulations, which may further affect the developments and metabolisms in humans via hormone mediations. The mechanisms of how CLEC18A involve in lipid synthesis pathways and hormone regulations need to be interpreted in the future works. |
