Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2005
Publication Date: 4/1/2006
Citation: Suderman, R.J., Dittmer, N.T., Kanost, M.R., Kramer, K.J., Beeman, R.W. 2006. Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols. Insect Biochemistry and Molecular Biology36: 353-365. Interpretive Summary: New insect pest control agents are needed to help control stored-product insect pests. We are investigating the biochemistry of insect cuticle tanning or sclerotization to identify novel target reactives that might be inhibited by insect growth regulators. In a collaborative project with biochemists at Kansas State University with support from the National Science Foundation, we demonstrated how proteins in the exoskeleton become linked to each other during tanning, which partly explains how the strength or hardness of the cuticle is established. This study is part of a long-term investigation of insect cuticle composition and the mechanism of stabilization, and has the long term goals of 1) synthesizing in the laboratory an artificial cuticle as a biomimetic material with an excellent strength to weight ratio and 2) identifying novel aspects of cuticle biochemistry and physiology that might be targeted by insect growth regulators and insect-selective insecticides.
Technical Abstract: We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-ß-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M. sexta pupal cuticular proteins MsCP36, MsCP20, and MsCP27 were expressed and purified to near homogeneity. Polyclonal antisera to these recombinant proteins recognized the native proteins in crude pharate brown-colored pupal cuticle homogenates. Furthermore, antisera to MsCP36, which contains a type-1 Rebers and Riddiford consensus sequence (RR-1), also recognized an immunoreactive protein in homogenates of larval head capsule exuviae, indicating the presence of an RR-1 cuticular protein in a very hard, sclerotized and nonpigmented cuticle. All three of the proteins formed small and large oligomers stable to boiling SDS treatment under reducing conditions after reaction with laccase and the N-acylcatecholamines. The optimal reaction conditions for MsCP36 polymerization were 0.3 mM MsCP36, 7.4 mM NBAD and 1.0 U/'l fungal laccase. Approximately 5–10% of the monomer reacted to yield insoluble oligomers and polymers during the reaction, and the monomer also became increasingly insoluble in SDS solution after reaction with the oxidized NBAD. When NADA was used instead of NBAD, less oligomer formation occurred, and most of the protein remained soluble. Radiolabeled NADA became covalently bound to the MsCP36 monomer and oligomers during cross-linking. Recombinant Manduca laccase (MsLac2) also catalyzed the polymerization of MsCP36.