Expression,Purification And Chitin-binding Mode Of CPR Family Cuticular Proteins Of Ostrinia Furnacalis

Asian corn borer(Ostrinia furnacalis)is the most important pest of corn in China.Understanding the mechanism of cuticle formation in O.furnacalis can help the biological control of this pest.Because cuticular protein and chitin are major components of insect cuticle,studying the interaction between them is crucial for elucidating the mechanism of insect cuticle formation.CP26703 and CP27370 are the most abundant cuticular protein gene of the integument and head capsule transcriptome data of O.furnacalis,respectively.Sequence analysis revealed that CP26703 and CP27370 belong to the RR-1 and RR-2 subfamily of CPR family cuticular protein,respectively.They both contain R&R domain,possibly function by binding to chitin.To study the interaction between the two cuticular proteins and chitin,this study carried out the following work:(1)Through analyzing the transcriptome data of head capsule and integument of O.furnacalis and comparing with NBCI database,cDNA sequences of cuticular protein genes CP26703 and CP27370 were obtained.Analysis of the amino acid sequence shows that the N-terminal of CP26703 and CP27370 have signal peptide sequences,which means they are secretory cuticular proteins.CP26703 belongs to hydrophilic protein,but CP27370 has a strongly hydrophobic sequence.Both proteins are composed of ?-sheet and random coil.Multiple sequence alignment shows that all CPR family cuticular proteins have highly conserved R&R sequence.(2)pET-SUMO vector was used to express SUMO-CP26703 and SUMO-CP27370,the soluble fusion protein.Then CP26703 and CP27370 were obtained by SUMO protease treatment and two steps of metal-chelating affinity chromatography with a yield of 5 mg/L and 6 mg/L,respectively.(3)Chitin binding assay revealed that the two cuticular proteins are selective for the binding to different types of chitin.CP26703 has strong binding ability to ?-chitin and deacetylated chitin,and has moderate binding ability to colloid chitin,but does not bind to ?-chitin.CP27370 generally has weak binding ability to these 4 kinds of chitins,but slightly stronger in binding to ?-chitin and deacetylated chitin.(4)The deacetylated chitin(chitosan)binding assay revealed that CP26703 can bind and induce the coacervation of deacetylated chitin,but CP27370 will not.As revealed by circular dichroism spectroscopy,after binding to deacetylated chitin,the proportions of parallel ?-sheet and ?-turn in of CP26703 increased by 18.4% and 11.7% respectively,whereas the proportions of anti-parallel ?-sheet and random coil decreased by 20.4% and 11.4% respectively.However,the proportions of anti-parallel ?-sheet and ?-turn in of CP27370 decreased by 10.0% and 6.3% respectively,whereas the proportions of parallel ?-sheet and random coil increased by 3.9% and 10.5% respectively.The results indicate that the two cuticular proteins will change their conformation to achieve better binding to chitin.At the same time,the addition of CP26703 protein increased the storage modulus(G ')of the deacetyl chitin solution by 2.4 times and the complex viscosity by 87.0%.The work revealed the two subfamily RR-1 and RR-2 belonging to CPR family cuticular proteins bind different forms of chitin with specificity and showed conformational changes during binding,which deepen the understanding of the interaction between cuticular proteins of CPR family and chitin and also provide further insights into the mechanism of insect cuticle formation.