Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/22/2002
Publication Date: 5/1/2003
Citation: SUDERMAN,R.J., ANDERSEN,S.O., HOPKINS,T.L., KANOST,M.R., KRAMER,K.J., CHARACTERIZATION AND CDNA CLONING OF THREE MAJOR PROTEINS FROM PHARATE PUPAL CUTICLE OF MANDUCA SEXTA, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 33: 331-343. 2003. Interpretive Summary: The insect exoskeleton or cuticle serves many functions including protection, locomotion, respiration and communication, and therefore must have very diverse mechanical and chemical properties to provide for optimization of each function. When an insect molts from the larval to the pupal stage, the new hard, tanned cuticle is very different in physical properties from the soft larval cuticle it replaces. This change must be due in part at least to differences in the proteins that make up the cuticular layers. Together with collaborators at Kansas State University and Copenhagen University, we describe in this paper the characterization of three abundant structural proteins and their genes, two of which are secreted by the epidermis into both the larval and pupal exoskeletons, and a third that is deposited only in the latter cuticle. These proteins are very difficult to isolate from their natural sources, but now recombinant forms of these proteins can be produced and used, together with other components of the exoskeleton, in future studies to prepare biomimetic materials for physical testing. These data are part of a detailed molecular characterization of newly discovered cuticular proteins/genes and will be utilized for understanding how these proteins become cross-linked during the sclerotization process. Also the results of this study provide new knowledge about insect support structures and their mechanisms of stabilization and regulation, and also yield new insights into how insects have evolved extracellular components with different properties, which contributed to their great diversity and success in nature. The information will be useful to scientists involved in the development of biotechnological methods of insect pest control, which are designed to disrupt insect cuticle physiology. Inhibition of this structural protein biochemistry would disrupt cuticle formation and prevent insect growth and development.
Technical Abstract: Three proteins, MsCP20, MsCP27 and MsCP36, that are secreted in greatest quantity into the pharate pupal cuticle of Manduca sexta (Hopkins et al., 2000) were purified and their amino acid sequences determined by mass spectrometry and Edman degradation. Although these proteins become sclerotized and insoluble in the pupal exoskeleton, their sequences contain features characteristic for proteins occurring in less sclerotized pliable cuticles, such as arthrodial membranes and soft larval cuticles. These proteins carry a secondary modification attached to a threonine residue, presumably an O-linked sugar moiety. cDNA clones of the genes for MsCP20, MsCP27 and MsCP36 were constructed from pharate pupal integument RNA. Close agreement was found between the amino acid sequences determined by Edman degradation and sequences deduced from the cDNA clones. The molecular masses determined by protein sequencing for MsCP20, 27, and 36 were 17713, 17448, and 29582 Da, respectively, in close agreement with the masses deduced from the corresponding cDNA clones (17711, 17410, and 29638 Da). Temporal expression analysis indicates that MsCP20 and MsCP36 transcripts are present throughout the fifth larval stadium, followed by a large increase in abundance prior to pupal ecdysis. MsCP27 was not detected during development of the fifth larval instar, but its transcript, like those of MsCP20 and 36, increased to a peak level just before pupal ecdysis. These results support the hypothesis that these proteins are synthesized by the epidermis and are subsequently deposited into the cuticle during the larval-pupal transformation of M. sexta where they become sclerotized in the formation of pupal exocuticle.