Characterization and cDNA cloning of three major proteins from pharate pupal cuticle of Manduca sexta

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, MsCP27, and MsCP36 were 17,713, 17,448, and 29,582 Da, respectively, in close agreement with the masses deduced from the corresponding cDNA clones (17,711, 17,410, and 29,638 Da). Temporal expression analysis indicates that MsCP20 and MsCP36 transcripts are present at low levels early in 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 MsCP36, increased to a peak level just before pupal ecdysis. Only the MsCP36 transcript was detected in adults. 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.