Cloning, identification and functional characterization of bovine free fatty acid receptor-1 (FFAR1/GPR40) in neutrophils.
Cloning, identification and functional characterization of bovine free fatty acid receptor-1 (FFAR1/GPR40) in neutrophils.
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DOI：
10.1371/journal.pone.0119715
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Hidalgo MA
中科院分区：
文献类型：
--
作者：
Manosalva C;Mena J;Velasquez Z;Colenso CK;Brauchi S;Burgos RA;Hidalgo MA
长链脂肪酸（LCFA）是G蛋白偶联受体FFAR 1（GPR 40）的配体，在分娩后奶牛血浆中增加，这是它们对传染病高度敏感的时期。本研究确定并分析了牛中性粒细胞中FFAR 1受体的功能作用，牛中性粒细胞是宿主抵御感染因子的第一道防线。我们从牛中性粒细胞中克隆了推定的FFAR 1受体，并分析了序列以构建同源模型。我们的结果显示，牛FFAR 1的序列与人FFAR 1的同源性为84%，与人FFAR 3/GPR 41的同源性为31%。因此，我们以人为模板构建了牛FFAR 1的同源模型。牛FFAR 1受体在中国仓鼠卵巢（CHO）-K1细胞中的表达增加了由LCFA、油酸（OA）和亚油酸（LA）诱导的细胞内钙水平;在短链脂肪酸丙酸的存在下，未观察到钙动员增加。此外，合成激动剂GW 9508增加CHO-K1/bFFAR 1细胞中的细胞内钙。OA和LA增加牛中性粒细胞内钙。此外，GW 1100（FFAR 1的拮抗剂）和U 73122（磷脂酶C（PLC）抑制剂）降低了CHO-K1/bFFAR 1细胞和中性粒细胞中FFAR 1配体诱导的细胞内钙。此外，抑制FFAR 1，PLC和PKC减少了FFAR 1配体诱导的基质金属蛋白酶（MMP）-9颗粒的释放和活性氧（ROS）的产生。因此，我们确定了牛FFAR 1受体，并证明了该受体在油酸或亚油酸激活的中性粒细胞中的功能作用。
Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.
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