The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide
The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide
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DOI：
10.1016/j.jprot.2013.05.022
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发表时间：
2013-07-11
影响因子：
3.3
通讯作者：
de Lima, Maria Elena
中科院分区：
文献类型：
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作者：
Cassoli, Juliana Silva;Verano-Braga, Thiago;de Lima, Maria Elena
海葵是有毒动物毒理学研究的热点之一，本工作的目的是通过生物化学技术的结合，寻找一种新的糖肽，并对其结构和功能进行研究。尽管先前的研究描述了海葵分泌物中存在的蛋白质化合物，但报道的一级序列的数量仍然很低。因此，为了了解S. duerdeni粘液，包括它们的生物学功能，一个强大的蛋白质组学方法与生物信息学工具一起使用。该方法也适用于其他海葵分泌物和动物毒液的分析。此外，新的肽结构可以出现有助于这些动物肽的多样性的知识。erization的化合物存在于分泌的海葵Stichodactyla duerdeni从巴西海岸。我们使用离线RPC-MALDI-TOF和在线nano-RPC-ESI-LTQ-Orbitrap蛋白质组学技术以及功能性生物测定的组合。粘液通过电刺激进行挤奶，并通过Sephadex G-50上的凝胶过滤进行分级，得到5个主要级分。将低分子量级分进一步进行RP-HPLC，得到35个新的亚级分，随后通过离线MALDI-TOF质谱法进行分析。MALDI肽质量指纹分析产生了多达134种不同的分子量，范围从m/z 901到10，833。在这些亚组分中，一个新的肽3431 Da，命名为U-SHTX-Sdd 1，纯化和完全测序的自动Edman降解和串联质谱。对U-SHTX-Sdd 1的分析揭示了在位置1处的修饰的O-HexNAc-Threatenin，据我们所知，其构成了报道的具有这种翻译后修饰的第一种海葵毒素。由于其序列与其他海葵毒素的相似性，U-SHTX-Sdd 1的药理活性进行了评估，通过电生理测量使用双电极电压钳技术克隆的电压门控钾通道亚型，表达在非洲爪蟾卵母细胞。然而，U-SHTX-Sdd 1在这些通道上没有显示出活性。本论文还利用大规模蛋白质组学方法对海葵化合物进行了研究，共鉴定出67种蛋白质和多肽。通过肽质量指纹图谱和高通量蛋白质组学分析来鉴定duerdeni分泌。此外，我们报告了一个新的糖肽的结构的生物化学技术的组合。尽管之前的研究描述了海葵分泌物中存在的蛋白质化合物，但报告的一级序列数量仍然很低。因此，为了了解S. duerdeni粘液，包括它们的生物学功能，一个强大的蛋白质组学方法与生物信息学工具一起使用。该方法也适用于其他海葵分泌物和动物毒液的分析。此外，新的肽结构可能会出现，有助于了解这些动物肽的多样性。(c)2013爱思唯尔有限公司版权所有。
Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional charactreport the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinforrnatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.erization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431 Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified.Biological significanceIn this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides. (c) 2013 Elsevier B.V. All rights reserved.