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.