Functional analysis of two lebocin-related proteins from Manduca sexta.
Functional analysis of two lebocin-related proteins from Manduca sexta.
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DOI：
10.1016/j.ibmb.2011.12.005
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发表时间：
2012-04
影响因子：
3.8
通讯作者：
Yu, Xiao-Qiang
中科院分区：
文献类型：
--
作者：
Rao, Xiang-Jun;Xu, Xiao-Xia;Yu, Xiao-Qiang
昆虫对微生物的感染会产生一组抗菌肽(AMPs)。大多数AMP是以非活性前体/前蛋白的形式合成的，需要蛋白水解性处理才能产生小的活性多肽。在这里，我们报道了烟草天牛Manduca sexta中两个Lebocin相关蛋白(Leb-B和Leb-C)的鉴定和功能分析。注射大肠杆菌、黄色微球菌和酿酒酵母后，幼虫脂肪体和血细胞中Leb-B和Leb-C的mRNA水平显著升高。用兔抗Leb-B多克隆抗体进行免疫印迹显示，注射微生物后，幼虫血淋巴中有大蛋白(S)和小肽(S)的积聚。这一结果和推导的氨基酸序列中RXXR基序的存在导致了我们的假设，即Leb-B/C可能是在幼虫血淋巴中加工产生短活性多肽的非活性前体。为了验证这一假设，我们表达并纯化了全长和各种片段的Leb-B和Leb-C作为硫氧还蛋白(Trx)融合蛋白。我们发现融合蛋白可以被诱导的幼虫血浆切割，并通过蛋白质测序确定了切割位置。多肽片段的抗菌活性也得到了验证，活性多肽位于Leb-B/C的N端，不同于位于C端附近的家蚕Lebocin 1-4。此外，我们还发现合成的Leb-B22-48肽不仅具有较高的抗菌活性，而且还能引起大肠杆菌细胞的凝集。我们的结果为研究鳞翅目昆虫中来波菌素前体的加工提供了有价值的信息。
Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1–4 that are located close to the C-termini. In addition, we found that synthetic Leb-B22–48 peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.
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影响因子：
2.9
作者：
Rao, Xiang-Jun;Yu, Xiao-Qiang
通讯作者：
Yu, Xiao-Qiang
影响因子：
4.8
作者：
Volz, J;Osta, MA;Müller, HM
通讯作者：
Müller, HM
影响因子：
2.9
作者：
Rayaprolu, Subrahmanyam;Wang, Yang;Kanost, Michael R.;Hartson, Steven;Jiang, Haobo
通讯作者：
Jiang, Haobo
影响因子：
--
作者：
Ganesan, Sandhya;Aggarwal, Kamna;Paquette, Nicholas;Silverman, Neal
通讯作者：
Silverman, Neal
DOI：
10.1006/bbrc.1997.7386
发表时间：
1997-09-29
影响因子：
3.1
作者：
Furukawa, S;Taniai, K;Yamakawa, M
通讯作者：
Yamakawa, M