Identification of novel CrylAc binding proteins in midgut membranes from Heliothis virescens using proteomic analyses
Identification of novel CrylAc binding proteins in midgut membranes from Heliothis virescens using proteomic analyses
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DOI：
10.1016/j.ibmb.2006.10.004
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发表时间：
2007-03-01
影响因子：
3.8
通讯作者：
Adang, Michael J.
中科院分区：
文献类型：
--
作者：
Krishnamoorthy, M.;Jurat-Fuentes, J. L.;Adang, Michael J.
氨基肽酶和碱性磷酸酶等蛋白质都是糖基磷脂酰肌醇(GPI)锚定蛋白，以前在绿色日光夜蛾中肠中被鉴定为CrylAc结合蛋白。为了鉴定更多的毒素结合蛋白，用磷脂酰肌醇磷脂酶C处理绿色隐翅虫幼虫的刷状缘膜囊泡，并用双向电泳法对释放的蛋白进行分离。选择与CrylAc结合能力较强的蛋白质斑点，用MALDI-TOF质谱仪结合多肽质量指纹图谱(PMF)和数据库检索进行鉴定。与以往的研究一样，绿色隐翅虫碱性磷酸酶被鉴定为CrylAc结合蛋白。V-ATP合成酶A亚基和肌动蛋白被鉴定为绿色隐翅虫中新的CrylAc结合蛋白。根据MS/MS裂解和从头测序预测了其他毒素结合蛋白，提供了用于数据库搜索的氨基酸序列，用于鉴定钙粘蛋白超家族的一个磷酸酶和一个可能的蛋白作为额外的CrylAc结合蛋白。(C)2006爱思唯尔有限公司。保留所有权利。
Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as CrylAc binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind CrylAc were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a CrylAc binding protein. V-ATP synthase subunit A and actin were identified as novel CrylAc binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional CrylAc binding proteins. (c) 2006 Elsevier Ltd. All rights reserved.