小白鼠子宮分泌蛋白 24p3 之生化研究

Abstract

24p3為小白鼠子宮所分泌之醣蛋白，由其蛋白質序列之比對發現與人類的中性球白明膠?結合蛋白（neutrophil gelatinase associated lipocalin, NGAL）極為相似，而兩者皆被認為是lipocalin家族的成員，可能負責某種尚未確定之疏水性小分子基質的傳送。由本研究室過去的研究而知24p3在子宮中的表現受到雌性素的誘導，然而在肝臟、腎臟及巨噬細胞中則分別被發現受到急性免疫反應、SV40感染及LPS等不同因素誘導而導致蛋白或mRNA產量增加，但24p3真正的生理功能仍待進一步探討。 本論文擬由不同途徑來探討24p3可能之生理意義。在基因層次的研究，我們嘗試決定24p3之基因結構序列，結果確定其基因包括6個exon及5個intron，長約3097個鹽基，此部分資料將可與NGAL比較以瞭解兩者之關係。然而我們並未能找到promoter部分之序列，因此無法瞭解24p3基因調控的機制。在蛋白細微構造的研究上，成功的移除24p3的氮端阻斷並確定其signal peptide的切割位置。同時我們亦確定24p3的2個半胱胺酸會形成1個雙硫鍵。這些細微構造與其他lipocalin，尤其是NGAL十分相近。在與基質結合的研究上，我們利用24p3的自身螢光來測定與疏水性小分子間的結合特性。結果顯示24p3與視黃醇、視黃酸、膽固油酯、油酸以及formyl-peptide等皆具有相似程度的結合能力。此一結果顯示24p3具有相當廣泛的基質結合特性，其基質結合專一性仍待進一步的研究。從圓二色光譜的分析中得知，24p3具有相當多的β結構，我們發現當膽固油脂與24p3結合後，會導致蛋白的部分β結構的破壞，顯示24p3的結構可能具有彈性，當基質與之結合時可能產生結構的改變。

24p3 is a secretory glycoprotein in mouse uterus. The protein sequence alignment manifests a very close relationship between 24p3 and human neutrophil gelatinase associated lipocalin (NGAL). These two proteins are also grouped into the lipocalin family and considered to be a transporter for an unknown small hydrophobic ligand. Our previous studies indicated that the expression of 24p3 in uterus is stimulated by oestrogen. On the other hand, other tissue like liver, kidney and macrophage had been reported to induce 24p3 in quite different manners, such as acute phase reaction, SV40 infection and LPS induction, respectively. Despite that the physiological function of 24p3 remains obscure. In this thesis, I tried to study 24p3 through different approaches. In the study of gene structure, I made an effort to sequence entire genomic structure of 24p3 and found that 24p3 have 6 exons and 5 introns with 3,097 bases. This data can be used in comparing with NGAL to find the relationship between them. Because fail to elucidate the promoter region of 24p3 gene thus unable to find clues for the mechanism of 24p3 expression. Meanwhile, I investigated protein fine structure. I de-blocked the N-terminal of 24p3 and established the signal peptide cleavage site. We also find there is one disulfite bond between the two cystein of 24p3. These fine structural features are quite similar to other lipocalins. In the presence of small lipophilic ligands, the fluorescence of 24p3 was perturbed. Analysis of the perturbant fluorescence revealed a single type of binding site of lipophilic ligand in the protein molecule. Retinol, retinoic acid, cholesteryl oleate, oleic acid and formyl peptide all show their binding affinity in the same order. Finally, the CD spectrum of 24p3 indicated a considerable amount of β form but less amount of helix in the protein molecule. Binding of cholesteryl oleate resulted in reduced the secondary structure to some extent, suggesting that the conformation of 24p3 is not rigid but rather flexible. The binding of ligand can induce the change in protein conformation.