Model reactions for insect cuticle sclerotization: participation of amino groups in the cross-linking of Manduca sexta cuticle protein MsCP36.
Current theories of sclerotization center on protein cross-linking and dehydration as major factors in the hardening and stability of the insect cuticle. Several studies have reported the identification of catechol-amino acid adducts from sclerotizing cuticle involving histidine, lysine, and tyrosine, though there have been no reports of a catechol linked between two amino acid residues. Previously, we reported an in vitro model system for sclerotization and observed that stable protein oligomers were formed, presumably through cross-links with oxidized catecholamines [Insect Biochem. Mol. Biol. (2006) 36, 353-365]. Using site-directed mutagenesis we created a mutant lacking histidine, rMsCP36(H65A/H178A), to investigate the possible involvement of the two histidine residues of MsCP36 in cross-linking. Surprisingly, this alteration had little or no effect on the formation of protein oligomers as determined by SDS-PAGE analysis. Blocking of the free amino groups in lysyl side chains and the amino-terminus by succinylation diminished, but did not eliminate, cross-linking of either rMsCP36 or rMsCP36(H65A/H178A). We also examined the possibility that cross-linking was due to intermolecular dityrosine linkages. Immunoblot analysis utilizing a monoclonal antibody known to recognize peptidyl dityrosine indicated that dityrosyl cross-links were present. Taken together, these results indicate that lysyl residues are important for the cross-linking of the cuticle protein rMsCP36, but that additional residues other than histidine can also contribute.
Suderman
Richard J
RJ
Department of Biochemistry, 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506, USA.
Dittmer
Neal T
NT
Kramer
Karl J
KJ
Kanost
Michael R
MR
D000595:Amino Acid Sequence; D000818:Animals; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D018402:Manduca / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D019476:Insect Proteins; C047663:cuticle proteins, insects