Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta.
Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta.
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DOI：
10.1016/j.dci.2010.01.008
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发表时间：
2010-06
影响因子：
2.9
通讯作者：
Jiang, Haobo
中科院分区：
文献类型：
--
作者：
Rayaprolu, Subrahmanyam;Wang, Yang;Kanost, Michael R.;Hartson, Steven;Jiang, Haobo
抗菌肽是昆虫天然免疫系统的重要组成部分。烟草天蛾中已鉴定出5种AMP，包括attacin、cecropin、moricin、gloverin和lebocin。在这里，我们报告的分离lebocin相关的cDNA克隆和抗菌活性的加工蛋白质产品。这17个cDNA序列由一个恒定的5′端和一个可变的3′端组成，3′端含有3个81个核苷酸的重复序列。从一株M. sexta基因组文库和Southern杂交结果表明该基因缺乏内含子，在基因组中以单拷贝存在。基因组序列包含13个完整的81个核苷酸重复和一个部分拷贝。北方印迹分析显示多个转录本具有主要的大小差异。M.注射细菌后，脂肪体中的sexta lebocin显著增加。蛋白质序列中的RXXR基序使我们假设前体被细胞内转化酶加工形成四种生物活性肽。为了验证这一假设，我们化学合成了这些肽并检查了它们的抗菌活性。肽1杀死革兰氏阳性和革兰氏阴性细菌。肽2，类似于大蜡螟AMP的序列，不影响细菌生长。肽3是无活性的，但在羧基末端具有额外的Arg的肽3对E.大肠杆菌在高的最低抑菌浓度。由81 bp重复序列编码的肽4在抗菌试验中无活性。前体蛋白的翻译后加工产生多种生物活性肽用于防御目的的假设是通过鉴定肽1，2，和3幼虫血淋巴通过液相色谱和串联质谱验证。与来自其他鳞翅目昆虫的直系同源物的比较表明，相同的机制可以用于从单个前体产生几个功能产物。
Antimicrobial peptides (AMPs) are a crucial component of the natural immune system in insects. Five types of AMPs have been identified in the tobacco hornworm Manduca sexta, including attacin, cecropin, moricin, gloverin, and lebocin. Here we report the isolation of lebocin-related cDNA clones and antibacterial activity of their processed protein products. The seventeen cDNA sequences are composed of a constant 5′ end and a variable 3′ region containing 3∼16 copies of an 81-nucleotide repeat. The sequence of the corresponding gene isolated from a M. sexta genomic library and Southern blotting results indicated that the gene lacks introns and exists as a single copy in the genome. The genomic sequence contained 13 complete and one partial copy of the 81-nucleotide repeat. Northern blot analysis revealed multiple transcripts with major size differences. The mRNA level of M. sexta lebocin increased substantially in fat body after larvae had been injected with bacteria. The RXXR motifs in the protein sequences led us to postulate that the precursors are processed by an intracellular convertase to form four bioactive peptides. To test this hypothesis, we chemically synthesized the peptides and examined their antibacterial activity. Peptide 1 killed Gram-positive and Gram-negative bacteria. Peptide 2, similar in sequence to a Galleria mellonella AMP, did not affect the bacterial growth. Peptide 3 was inactive but peptide 3 with an extra Arg at the carboxyl terminus was active against E. coli at a high minimum inhibitory concentration. Peptide 4, encoded by the 81-bp repeat, was inactive in the antibacterial tests. The hypothesis that posttranslational processing of the precursor proteins produces multiple bioactive peptides for defense purposes was validated by identification of peptides 1, 2, and 3 from larval hemolymph via liquid chromatography and tandem mass spectrometry. Comparison with the orthologs from other lepidopteran insects indicates that the same mechanism may be used to generate several functional products from a single precursor.
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影响因子：
2.2
作者：
LADENDORFF, NE;KANOST, MR
通讯作者：
KANOST, MR
DOI：
10.1073/pnas.89.22.10915
发表时间：
1992-11-15
影响因子：
11.1
作者：
HENIKOFF, S;HENIKOFF, JG
通讯作者：
HENIKOFF, JG
影响因子：
56.9
作者：
Christophides, GK;Zdobnov, E;Kafatos, FC
通讯作者：
Kafatos, FC
影响因子：
3.4
作者：
DUNN, PE;DRAKE, DR
通讯作者：
DRAKE, DR
影响因子：
14.9
作者：
Chenna, R;Sugawara, H;Thompson, JD
通讯作者：
Thompson, JD