Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology.
Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology.
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DOI：
10.1371/journal.pone.0164593
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Peterson JM
中科院分区：
文献类型：
--
作者：
Li Y;Ozment T;Wright GL;Peterson JM
C1q TNF相关蛋白3（CTRP3）是分泌蛋白家族的成员，发挥多种生物学作用。我们最初的工作确定了CTRP 3作为非酒精性脂肪肝（NAFLD）有效治疗的前景。具体地，我们证明了喂食高脂肪饮食的小鼠在用CTRP 3治疗时没有发展成NAFLD。本项目的目的是鉴定介导CTRP3肝脏作用的假定受体。我们使用配体-受体糖捕获技术和基于TriCEPS ™的配体-受体捕获（LRC-TriCEPS; Dualsystems Biotech AG）。在H4IIE大鼠肝癌细胞系上进行以CTRP3-FLAG蛋白作为配体和胰岛素作为对照配体的LRC-TriCEPS实验。初步分析证明了TriCEPS与CTRP 3的有效偶联。此外，流式细胞术分析（FACS）证明了CTRP 3-TriCEPS和胰岛素-TriCEPS复合物与细胞表面聚糖的成功氧化和交联。在对照数据集中鉴定了胰岛素受体，证明了TriCEPS在这些条件下的实用性。在CTRP 3处理的细胞中，观察到总共富集了261种肽。从这些实验中鉴定了5种CTRP 3的推定受体，其中两种达到统计学显著性：溶酶体相关膜蛋白1（LAMP-1）和溶酶体膜蛋白2（LIMP II）。后续免疫共沉淀分析证实了LAMP 1和CTRP 3之间的关联，并且使用多克隆抗体阻断LAMP 1的潜在结合位点的进一步测试阻止了CTRP 3与细胞的结合。LRC-TriCEPS方法成功鉴定了CTRP3的潜在新型受体。CTRP3受体的鉴定是开发目前不存在的用于治疗NAFLD的小分子候选药物的重要先决条件。
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.
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溶酶体膜的糖蛋白。
影响因子：
7.8
作者：
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通讯作者：
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4.8
作者：
Crombie, R;Silverstein, R
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