Proteomic Analysis of Insect Molting Fluid with a Focus on Enzymes Involved in Chitin Degradation

Cuticular chitin degradation is extremely
important for insect
growth and development, which has not been fully understood thus far.
One obstacle to understanding this mechanism is the lack of a systematic
analysis of the chitinolytic enzymes involved in cuticular chitin
degradation. In this study, we used the silkmoth Bombyx mori as a model organism and compared proteomic analyses for larval-pupal
(L-P) and pupal-adult (P-A) molting fluids using tandem mass tag quantitative
mass spectrometry. There were 195 proteins identified from both L-P
and P-A molting fluids. A total of 170 out of 195 proteins were deduced
to be secretory and were enriched for GO terms associated with chitin
metabolism and proteolysis by using AgriGO. Although the chitinolytic
enzymes are encoded by many insect genes, the proteomics analysis
unexpectedly showed that only four chitinolytic enzymes with the combination
“211” were abundant in both molting fluids, namely,
two insect GH18 Chitinase family members (ChtI and ChtII), one bacterial-type
GH18 Chitinase (Chi-h), and one insect GH20 hexosaminidase (Hex1).
A tissue-specific and stage-specific gene expression pattern verified
that the “211” enzymes are involved in cuticular chitin
degradation. This work first demonstrates that specific enzymes ChtI,
ChtII, Chi-h, and Hex1 can be assigned to cuticular chitin degradation.

角质层几丁质降解对于昆虫的生长发育至关重要，然而，这一机制至今尚未得到充分理解。理解该机制的障碍之一在于缺乏对参与角质层几丁质降解的几丁质酶的系统分析。在本研究中，我们以家蚕 Bombyx mori 作为模式生物，利用串联质谱定量质谱分析了幼虫-蛹期（L-P）和蛹-成虫期（P-A）蜕皮液。从L-P和P-A蜕皮液中鉴定出195种蛋白质。其中，170种蛋白质被推断为分泌型，并经AgriGO分析富集了与几丁质代谢和蛋白质分解相关的GO术语。尽管许多昆虫基因编码了几丁质酶，但蛋白质组学分析意外地显示，只有四种具有“211”组合的几丁质酶在两种蜕皮液中含量丰富，分别是两种昆虫GH18几丁质酶家族成员（ChtI和ChtII）、一种细菌型GH18几丁质酶（Chi-h）和一种昆虫GH20己糖胺酶（Hex1）。特定的组织特异性和阶段特异性基因表达模式证实了“211”酶参与角质层几丁质降解。本研究首次证明了特定的酶ChtI、ChtII、Chi-h和Hex1可以被分配到角质层几丁质降解过程中。