Tissue Transglutaminase Is a Negative Regulator of Monomeric Lacritin Bioactivity
Tissue Transglutaminase Is a Negative Regulator of Monomeric Lacritin Bioactivity
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DOI：
10.1167/iovs.12-11488
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发表时间：
2013-03-01
影响因子：
4.4
通讯作者：
Laurie, Gordon W.
中科院分区：
文献类型：
--
作者：
Velez, Francisco V.;Romano, Jeffrey A.;Laurie, Gordon W.
目的.生物活性液体的摩尔计数可以在日益增长的蛋白质组学关注上皮生物学中暴露新的调节机制。对于眼睛表面上皮细胞的活力和正常视力至关重要的是薄但富含蛋白质的泪膜，其中小的泪液糖蛋白lacritin似乎发挥突出的促分泌、细胞保护和促有丝分裂作用。虽然在细胞培养物中的最佳生物活性水平是1至10 nM的双相剂量最佳值，ELISA表明在健康成年人中持续的泪液催乳素浓度在中微摩尔范围内。在这里，我们确定了一个和解机制。制备了抗lacritin 1F 5单克隆抗体，并与一种新的抗C-末端多克隆抗体一起应用于泪液和组织Western印迹。在体外组织转氨酶（Tgm 2）的交联进行监测，并通过质谱表征。在人泪液或唾液中对催泪蛋白的印迹令人惊讶地检测到具有较高分子量的免疫反应性物质，并且显著性等于或超过单体糖基化催泪蛋白的类似23至25 kDa条带。外源性Tgm 2在1分钟内启动了lacritin交联，并在90分钟内完成-即使只有0.1 nM lacritin，并涉及供体赖氨酸82和85和受体谷氨酰胺106的syndecan-1结合结构域。催乳素掺入催乳素耗尽的泪液中形成多聚体，与泪液中的0.6 μ M TGM 2相似。当Tgm 2失活时，交联不存在，并且与重组单体不同，交联的lacritin与syndecan-1的结合较差。由于多配体蛋白聚糖-1结合是催泪蛋白促有丝分裂和细胞保护活性所必需的，因此TGM 2交联负调节催泪蛋白生物活性。（Invest Ophthalmol维斯科学。2013; 54：2123-2132）DOI：10.1167/iovs.12-11488
PURPOSE. Molar accounting of bioactive fluids can expose new regulatory mechanisms in the growing proteomic focus on epithelial biology. Essential for the viability of the surface epithelium of the eye and for normal vision is the thin, but protein-rich, tear film in which the small tear glycoprotein lacritin appears to play a prominent prosecretory, cytoprotective, and mitogenic role. Although optimal bioactive levels in cell culture are 1 to 10 nM over a biphasic dose optimum, ELISA suggests a sustained tear lacritin concentration in the midmicromolar range in healthy adults. Here we identify a reconciling mechanism.METHODS. Monoclonal anti-lacritin 1F5 antibody was generated, and applied together with a new anti-C-terminal polyclonal antibody to tear and tissue Western blotting. In vitro tissue transglutaminase (Tgm2) cross-linking was monitored and characterized by mass spectrometry.RESULTS. Blotting for lacritin in human tears or saliva surprisingly detected immunoreactive material with a higher molecular weight and prominence equal or exceeding the similar to 23 to 25 kDa band of monomeric glycosylated lacritin. Exogenous Tgm2 initiated lacritin cross-linking within 1 minute and was complete by 90 minutes-even with as little as 0.1 nM lacritin, and involved the donors lysine 82 and 85 and the acceptor glutamine 106 in the syndecan-1 binding domain. Lacritin spiked into lacritin-depleted tears formed multimers, in keeping with similar to 0.6 mu M TGM2 in tears. Cross-linking was absent when Tgm2 was inactive, and cross-linked lacritin, unlike recombinant monomer, bound syndecan-1 poorly.CONCLUSIONS. Since syndecan-1 binding is necessary for lacritin mitogenic and cytoprotective activities, TGM2 cross-linking negatively regulates lacritin bioactivity. (Invest Ophthalmol Vis Sci. 2013; 54: 2123-2132) DOI: 10.1167/iovs.12-11488