Protein cross-linking Reactions for Insect Cuticle Sclerotization

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This project will address how insects use structural proteins, catechols, oxidative enzymes, and chitin to stabilize their exoskeletons. The exoskeleton is primarily composed of two structural polymers, protein and chitin. The cuticle proteins are cross-linked by quinones derived from catechols by a process called sclerotization, a vital metabolic step that occurs during each stage of insect development. This interdisciplinary project will investigate the interactions of quinonoid metabolites of catecholic precursors with cuticular proteins from the tobacco hornworm, Manduca sexta. The research will test a hypothesis that sclerotization involves the formation of carbon-nitrogen cross-links between the nucleophilic imidazole nitrogens of histidine and the ring or side-chain carbons of o-quinones and p-quinone methides derived from catechols. A second hypothesis to be tested is that differences in hardness and flexibility from distinct regions of an exoskeleton are due to the occurrence of different sets of proteins and cross-linking agents expressed by the underlying epidermis. The first objective is to identify and characterize proteins expressed in cuticles with different physical properties. The cuticle of the M. sexta larval abdomen is soft and flexible, whereas the pupal abdomen is rigid and the larval head capsule quite hard. cDNAs obtained for proteins that are expressed in pupal abdominal cuticles, larval abdominal cuticle, and head capsule will be used to study the pattern of expression of the corresponding genes with regard to development and anatomy. The second objective is to investigate the structure and properties of two M. sexta laccases. Laccases oxidize catechols to quinones or quinone methides. Although laccases have been proposed to be important in insect cuticle sclerotization, they have received little study. cDNAs for two M. sexta laccases have been isolated, and their expression patterns characterized. The insect laccases are composed of a catalytic domain similar to those of fungal and plant laccases, as well as a unique amino-terminal domain not present in other known proteins. M. sexta laccases 1 and 2 will be expressed as recombinant proteins for characterization of their enzymatic properties, investigation of the function of the amino-terminal and catalytic domains, and for use in model sclerotization reactions described in objective 3. The third objective is to investigate biochemical and biophysical properties of cuticle using model sclerotization reactions. Selected proteins hypothesized to be responsible for formation of rigid or flexible cuticle will be expressed as recombinant proteins and used in model sclerotization reactions including a catechol, a phenoloxidase, and in the presence or absence of chitin or a chitin derivative. Protein-protein cross-links, protein-carbohydrate cross-links, and the effects of different catechols and cuticle proteins on the physical properties of cross-linked protein hydrogels and chitin-protein films produced in vitro will be investigated and compared with cuticles from different anatomical regions of M. sexta. Site directed mutagenesis experiments will test a hypothesis that histidine residues are involved in protein cross-linking during sclerotization. The exoskeleton of insects is composed of cuticle proteins and a polysaccharide called chitin. Each time an insect molts, these components of the exoskeleton are synthesized and then they are chemically cross-linked in a process called sclerotization. Sclerotization results in a light-weight material with unusual strength. This project investigates the chemistry of sclerotization, which is not yet well understood. Model sclerotization reactions will be carried out to determine how the cuticle proteins are linked to each other and to chitin. The biochemistry of insect cuticle sclerotization may be a useful target for design of novel methods for controlling insect pests of agriculture and insects that are vectors of diseases. New types of cross-linked biopolymers, based on the chemistry of cuticle sclerotization, may have potential uses in medicine and industry.