Regulation of Protein Cross-Linking Reactions for Insect Cuticle Sclerotization

Kansas State University, Manhattan KS

Investigators

Abstract

Insect cuticle sclerotization or tanning is a vital process that occurs during each stage of development to harden and stabilize the newly secreted cuticular exoskeleton. The exoskeleton serves many functions including protection, locomotion, respiration and communication, and has unique mechanical and chemical properties to optimize each function. The structural polymers, protein and chitin, make up the bulk of the cuticle. Chemical interactions of one or both of these polymers with quinonoid tanning agents are largely responsible for the final properties of the exoskeleton. We wish to investigate the reactions which help to stabilize the pupal cuticle of the tobacco hornworm, Manduca sexta, our model insect. The main hypothesis for sclerotization involves the formation of carbon-nitrogen bonds to cross-link histidine side chains in cuticular proteins to the ring or side-chain carbons of o-quinones and p-quinone methides derived from catechols. This research will address how insects use structural proteins, catechols, and oxidative enzymes to stabilize their exoskeletons. The first objective of this project is to identify directly the naturally occurring chemicals in cuticle, which crosslink structural proteins. The second objective is to further characterize a tanning phenoloxidase, laccase, which catalyzes quinonoid formation in the cuticle during sclerotization. We will investigate the regulation of expression of the laccase gene and the localization of laccase in the integument. The third objective will be to clone the cDNAs for three of the major cuticular proteins that become cross-linked during sclerotization, express these as recombinant proteins, and use them for in vitro model cuticle reconstitution experiments, where, together with the catechols, the recombinant phenoloxidases should generate proteins cross-linked via catechols. These studies of the oxidative conjugation of catechols and proteins in the insect exoskeleton will provide new fundamental information about the natural cross-linking agents and their covalent interactions with cuticular proteins. Understanding how this metabolic biochemistry facilitates cuticle sclerotization via protein cross-linking is of vital importance to insect biology. Specific interference with this process would be detrimental to insect growth and development, and also may facilitate the development of new approaches to selective insect pest control.