分泌型唾液酸受體其生成機制與抑制骨髓細胞發炎反應的功能探討

Soluble Siglec-14 is generated by alternative splicing and suppresses myeloid inflammatory responses

黃柏鈞(Po-Chun Huang)

指導教授 ： 安形高志

國立臺灣大學/生命科學院/生化科學研究所/博士(2018年)

https://doi.org/10.6342/NTU201803214

摘要

人類唾液酸結合蛋白14 (Siglec-14)為唾液酸結合蛋白家族成員之一，其具有增強細菌誘導的髓細胞促發炎反應。在臨床研究病例中，Siglec-14蛋白已被證實具有影響某些細菌誘導的反應。雖然Siglec-14蛋白已知屬於第一型膜蛋白，但是研究發現人類血液中存在著分泌型Siglec-14蛋白。然而，對於分泌型Siglec-14蛋白產生的機制與其存在人體內所扮演的角色，其研究所知甚少。本研究的目的著重於探索分泌型Siglec-14蛋白如何產生與其在人體內的功能。
我發現Siglec-14蛋白具有兩種傳訊RNA剪接變異體，分別為具備或是缺少內含子5的變異體。由於內含子5帶有終止密碼子，造成蛋白轉譯提前終止。缺乏內含子5的Siglec-14，其外顯子6可以被轉譯形成跨膜部位並組成第一型膜蛋白。藉由專一性辨認分泌型Siglec-14之C端胜肽片段的抗體，實驗證實由分泌型Siglec-14傳訊RNA剪接異變體所轉譯的蛋白是存在於人類血清中的。
在膜型Siglec-14蛋白表現的髓細胞實驗中，我分析重組分泌型Siglec-14蛋白對於促發炎細胞激素的影響。結果發現分泌型Siglec-14蛋白抑制無莢膜流感嗜血桿菌引起的髓細胞發炎反應並呈現劑量依賴性的狀況。此結果可能係由分泌型Siglec-14蛋白阻礙膜型Siglec-14與類鐸受體2 (Toll-like receptor 2)之間在細胞表面上的交互作用。內含子5的一部分序列會形成一種RNA二級結構名為G-四聯體，它可能涉及內含子5剪接的效率。綜上所述，分泌型Siglec-14蛋白在膜型Siglec-14蛋白引發的促發炎反應中扮演著負向調控的角色。

關鍵字

唾液酸結合蛋白14 ； 分泌型唾液酸結合蛋白 ； 選擇性剪接 ； 無莢膜流感嗜血桿菌 ； 類鐸受體2 ； G-四聯體

並列摘要

Human Siglec-14 is a member of the Siglec family of sialic acid-binding proteins. Siglec-14 enhances anti-bacterial myeloid cell pro-inflammatory responses. In some clinical conditions, Siglec-14 was shown to influence some bacterially induced responses in patients. Although Siglec-14 is a type 1 transmembrane protein, a soluble form of Siglec-14 was also found in human serum. However, the mechanism how soluble Siglec-14 is generated and what role it plays remain unknown. In this study, I aimed to understand how the soluble Siglec-14 is generated and what the function of soluble Siglec-14 is.
I found two major mRNA splice variants that either contain or lack intron 5, which contains an in-frame stop codon. The variant containing intron 5 yields soluble form by premature termination (as the exon 6 encodes transmembrane domain of Siglec-14), while the one without intron 5 yields the transmembrane form. I prepared antibodies against the C-terminal peptide segment unique to the soluble form of Siglec-14, and confirmed that the soluble form in human serum is indeed generated by the splice variant I found.
I analyzed the influence of recombinant soluble Siglec-14 on pro-inflammatory cytokine production by membrane-bound Siglec-14 (mSiglec-14)-expressing myeloid cells. Soluble Siglec-14 suppressed non-typeable Haemophilus influenzae (NTHi)-stimulated response in a dose-dependent manner, which may be the result of interrupting the interaction between mSiglec-14 and toll-like receptor 2 on cell surface. A part of the intron 5 assumes an RNA secondary structure called G-quadruplex, which may be involved in the regulation of intron 5 splicing. In summary, I propose that soluble Siglec-14 is a negative regulator for the pro-inflammatory responses triggered by transmembrane Siglec-14.

並列關鍵字

Siglec-14 ； soluble Siglec ； alternative splicing ； non-typeable Haemophilus influenzae ； Toll-like receptor 2 ； G-quadruplex

參考文獻

1. Crocker, P.R., J.C. Paulson, and A. Varki, Siglecs and their roles in the immune system. Nat Rev Immunol, 2007. 7(4): p. 255-66.

2. Varki, A. and T. Angata, Siglecs--the major subfamily of I-type lectins. Glycobiology, 2006. 16(1): p. 1R-27R.

3. Properties and distribution of a lectin-like hemagglutinin differentially expressed by murine stromal tissue macrophages. The Journal of Experimental Medicine, 1986. 164(6): p. 1862-1875.

4. Stamenkovic, I. and B. Seed, The B-cell antigen CD22 mediates monocyte and erythrocyte adhesion. Nature, 1990. 345: p. 74.

5. Miescher, G.C., et al., Reciprocal expression of myelin-associated glycoprotein splice variants in the adult human peripheral and central nervous systems1The nucleotide sequence of the human S-MAG cDNA fragment has been made available in the EMBL Nucleotide Sequence Database under the accession number: X98405.1. Molecular Brain Research, 1997. 52(2): p. 299-306.