Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx mori
Analysis of expression and chitin-binding activity of the wing disc cuticle protein BmWCP4 in the silkworm, Bombyx mori
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家蚕翅盘角质层蛋白 BmWCP4 的表达及几丁质结合活性分析
DOI：
10.1111/1744-7917.12231
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发表时间：
2016-12-01
期刊：
影响因子：
4
通讯作者：
Feng, Qi-Li
中科院分区：
文献类型：
--
作者：
Deng, Hui-Min;Li, Yong;Feng, Qi-Li
昆虫的外骨骼主要由甲壳素细丝和角质层蛋白质连接而成。当昆虫蜕皮时，外骨骼的角质层通过降解旧的几丁质和角质层蛋白并合成新的来更新。本文研究了家蚕翅盘角质层蛋白BmWCP4的几丁质结合活性。序列分析表明，该蛋白具有一个保守的亲水性的"R & R"几丁质结合结构域（CBD）。Western blotting结果表明，BmWCP 4主要表达在翅盘的表皮中的晚期徘徊和早期蛹阶段。免疫组化结果显示，在游行期第2天，BmWCP4主要表达于含翅芽和气管原始细胞的翅盘组织中。分别制备并纯化了重组全长BmWCP4蛋白、"R & R" CBD肽（CBD）、非CBD肽（BmWCP4-CBD-）、四个单位点突变肽（M-1、M-2、M-3和M-4）和四位点突变肽（M-F），用于体外几丁质结合试验。结果表明，全长蛋白和"R & R" CBD肽都能与几丁质结合，而BmWCP 4-CBD-不能与几丁质结合。单残基突变体M-1、M-2、M-3和M-4的几丁质结合活性有所降低，但没有完全丧失，而四位点突变的MF则完全丧失了几丁质结合活性。这些数据表明，BmWCP（4）蛋白在幼虫向蛹转化过程中通过结合翅中的几丁质丝起关键作用。保守的芳香族氨基酸在几丁质和角质层蛋白之间的相互作用中是关键的。
The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic "R&R" chitin-binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, "R&R" CBD peptide (CBD), non-CBD peptide (BmWCP4-CBD-), four single site-directed mutated peptides (M-1, M-2, M-3 and M-4) and four-sites-mutated peptide (M-F) were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the "R&R" CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M-1, M-2, M-3 and M-4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP(4) protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.