Presentation Information
[P04-492]Comparison of chitin-binding affinity of CPR proteins at different stages of expression during the elytra formation of the Japanese rhinoceros beetle
○Kodai Kimura1, Satoshi Murata1, David Kisailus2,3, Atsushi Arakaki1 (1. Graduate School of Engineering, Tokyo University of Agriculture and Technology (Japan), 2. Department of Materials Science and Engineering, University of California Irvine (USA), 3. Materials and Manufacturing Technologies Program, University of California Irvine (USA))
Keywords:
Cuticular proteins (CPs),Beetle,Chitin-binding,Bio-inspired materials
[Purpose]
Beetle cuticles achieve both light weight and high strength by proteins that control the orientation of chitin fibers to form a highly ordered layered structure. This biodegradable structure is attracting attention as an environmentally friendly functional material, and elucidating its formation mechanism is highly anticipated. CPR proteins, the most abundant in the cuticle, possess a chitin-binding domain and are involved in cuticle structure formation. Our laboratory has identified three distinct protein groups in the matured cuticle of the Japanese rhinoceros beetle, Trypoxylus dichotomus, differing in expression timing and amino acid sequence. Since these proteins are expressed during different stages of cuticle structure formation, they are hypothesized to have distinct functions. Because chitin-binding affinity enables the assembly of chitin and its interface with matrix proteins, which play a critical role in the macroscopic mechanical properties of the cuticle, it is inferred that these groups possess varying binding affinities. Therefore, this study quantitatively compared the chitin-binding abilities of proteins from three distinct groups, aiming to elucidate their respective roles in the cuticle formation mechanism.
[Method]
Using cDNA prepared from Trypoxylus dichotomus, three proteins—CPR41, CPR79, and CPR83—representing the different groups expressed at different times were produced via an E. coli expression system. For the chitin-binding assay, each CPR protein was incubated with chitin for one hour. The non-binding fraction was collected via centrifugation, while the binding fraction was recovered by dissociating the proteins using SDS and boiling. Protein concentrations were quantified using the BCA assay to evaluate binding affinity.
[Results]
The binding assays successfully demonstrated clear differences among the three proteins. CPR83 exhibited the highest chitin-binding affinity, followed by CPR79, and CPR41 (which showed the lowest binding capacity). Furthermore, a detailed comparison of their amino acid sequences revealed a strong correlation between protein composition and binding strength; specifically, an increased number of aromatic and polar amino acids within the proteins was positively correlated with a higher chitin-binding capacity.
[Consideration]
The results suggest that π-CH interactions between aromatic residues and chitin, alongside hydrogen bonding with polar residues, may contribute to binding stability. Notably, CPR41 is highly expressed during the immature stage, CPR79 during the maturation stage, and CPR83 during the late maturation stage. This sequential increase in chitin-binding affinity suggests a mechanism where the gradual reinforcement of the chitin-protein matrix is essential for enhancing the structural stability of the developing elytra.
[Conclusion]
This study demonstrated that the three CPR protein groups possess distinct chitin-binding affinities corresponding to their temporal expression patterns. This gradual shift in binding strength likely drives the chitin assembly and interfacial bonding between chitin and matrix proteins during maturation of the beetle cuticle, providing a blueprint for designing biomimetic materials with tunable mechanical properties.
Beetle cuticles achieve both light weight and high strength by proteins that control the orientation of chitin fibers to form a highly ordered layered structure. This biodegradable structure is attracting attention as an environmentally friendly functional material, and elucidating its formation mechanism is highly anticipated. CPR proteins, the most abundant in the cuticle, possess a chitin-binding domain and are involved in cuticle structure formation. Our laboratory has identified three distinct protein groups in the matured cuticle of the Japanese rhinoceros beetle, Trypoxylus dichotomus, differing in expression timing and amino acid sequence. Since these proteins are expressed during different stages of cuticle structure formation, they are hypothesized to have distinct functions. Because chitin-binding affinity enables the assembly of chitin and its interface with matrix proteins, which play a critical role in the macroscopic mechanical properties of the cuticle, it is inferred that these groups possess varying binding affinities. Therefore, this study quantitatively compared the chitin-binding abilities of proteins from three distinct groups, aiming to elucidate their respective roles in the cuticle formation mechanism.
[Method]
Using cDNA prepared from Trypoxylus dichotomus, three proteins—CPR41, CPR79, and CPR83—representing the different groups expressed at different times were produced via an E. coli expression system. For the chitin-binding assay, each CPR protein was incubated with chitin for one hour. The non-binding fraction was collected via centrifugation, while the binding fraction was recovered by dissociating the proteins using SDS and boiling. Protein concentrations were quantified using the BCA assay to evaluate binding affinity.
[Results]
The binding assays successfully demonstrated clear differences among the three proteins. CPR83 exhibited the highest chitin-binding affinity, followed by CPR79, and CPR41 (which showed the lowest binding capacity). Furthermore, a detailed comparison of their amino acid sequences revealed a strong correlation between protein composition and binding strength; specifically, an increased number of aromatic and polar amino acids within the proteins was positively correlated with a higher chitin-binding capacity.
[Consideration]
The results suggest that π-CH interactions between aromatic residues and chitin, alongside hydrogen bonding with polar residues, may contribute to binding stability. Notably, CPR41 is highly expressed during the immature stage, CPR79 during the maturation stage, and CPR83 during the late maturation stage. This sequential increase in chitin-binding affinity suggests a mechanism where the gradual reinforcement of the chitin-protein matrix is essential for enhancing the structural stability of the developing elytra.
[Conclusion]
This study demonstrated that the three CPR protein groups possess distinct chitin-binding affinities corresponding to their temporal expression patterns. This gradual shift in binding strength likely drives the chitin assembly and interfacial bonding between chitin and matrix proteins during maturation of the beetle cuticle, providing a blueprint for designing biomimetic materials with tunable mechanical properties.
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