Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.
Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.
复制标题
检测非灵长类泪液中主要作为 C 末端片段的促分泌性乳泌素。
DOI：
10.1167/iovs.11-8567
复制
发表时间：
2012
影响因子：
4.4
通讯作者：
Laurie,GordonW
中科院分区：
文献类型：
--
作者：
Laurie,DianeE;Splan,RebeccaK;Green,Kari;Still,KatherineM;McKown,RobertL;Laurie,GordonW
目的：催泪素是一种人泪液糖蛋白，可促进培养的大鼠泪腺泡细胞的基础泪液蛋白分泌和亚融合的人角膜上皮细胞的增殖。当局部添加到兔眼，催泪素促进基础流泪。尽管这些活动对几个物种，lacritin的存在，在非灵长类动物的眼泪或其他组织尚未探讨。在这里，我们探测了正常马泪液中的催泪蛋白。从人LACRT基因与高质量草图马基因组（EquCab 2. 0)和分析收集正常马泪液并通过Western印迹、ELISA和质谱法进行分析。采用新产生的兔抗体，分别针对人催乳素的N-和C-末端区域。同一性分别为75%和45%，在核苷酸和蛋白质水平。结构特征是保守的，包括C-末端两亲性α-螺旋。抗C-末端抗体在马眼泪中强烈检测到一条13 kDa的条带，该条带通过质谱法进行了验证。在人类泪液中，相同的抗体检测到未切割的催乳素（1024 kDa）强烈和C-末端片段的1013和1011 kDa弱。抗-N-末端抗体与24 kDa马抗原轻微反应，与抗-C-末端反应性213 kDa物质无反应。通过ELISA，马C-末端与N-末端免疫反应性的相应水平相似。催泪蛋白存在于马泪液中，主要作为与人催泪蛋白中的促有丝分裂和杀菌区域同源的C-末端片段，表明在角膜伤口修复中的潜在益处。
Purpose.: Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears.Methods.: Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2. 0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N-and C-terminal regions of human lacritin were employed.Results.: Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a∼ 13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼ 24 kDa) strongly and C-terminal fragments of∼ 13 and∼ 11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a∼ 24 kDa horse antigen and showed no reaction with the anti-C-terminal–reactive∼ 13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA.Conclusions.: Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.