Insect Biochemistry and Molecular Biology 30 (2000) 19–27 www.elsevier.com/locate/ibmb Pupal cuticle proteins of Manduca sexta: characterization and profiles during sclerotization Theodore L. Hopkins a,* , L. John Krchma a , Saad A. Ahmad a , Karl J. Kramer b a Department of Entomology, Waters Hall, Kansas State University, Manhattan, KS 66506-4004, USA b Grain Marketing and Production Research Laboratory, ARS-USDA, Manhattan, KS 66502, USA Received 17 May 1999; received in revised form 9 August 1999; accepted 11 August 1999 Abstract Proteins in pupal abdominal cuticle of the tobacco hornworm, Manduca sexta, were characterized during the pre-ecdysial and post-ecdysial periods of sclerotization and endocuticle formation. Protein extractability decreased dramatically as the cuticle became sclerotized through 6 h post-ecdysis, but increased rapidly from 9 to 48 h as endocuticular layers were secreted. Nearly 100 proteins that were extracted from pre-ecdysial cuticle became largely insoluble during sclerotization. Three major proteins in this group destined to become exocuticle had apparent molecular masses (Mapp) of 20, 27 and 36 kDa, and were designated MS-PCP20, MS- PCP27, and MS-PCP36. Amino acid analysis revealed glycine to predominate in all three proteins, and alanine, aspartate, glutamate, proline and serine were also relatively abundant. Histidine residues, which provide sites for adduct and cross-link formation with quinone metabolites of N-β-alanyldopamine during sclerotization of pupal cuticle, ranged from 2 to 3 mol %. N-Terminal amino acid analysis of MSPC-20 and MSPC-36 also revealed some sequence similarities indicating they may be related. An almost entirely new group of proteins appeared by 9 h as endocuticule secretion began, and these increased in abundance through 48 h post- ecdysis. Two of these were major proteins with Mapps of 33 and 34 kDa, and they also had close similarities in their N-terminal amino acid sequences. This study showed that the large number of proteins secreted into the presumptive exocuticle of the pupa before ecdysis are involved in sclerotization reactions and as a consequence become largly insoluble. The epidermis then switches to the secretion of an entirely new group of proteins that are involved in formation of the endocuticle.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Insect cuticle; Cuticular proteins; Cuticle tanning; Sclerotization; Tobacco hornworm; Manduca sexta; Catecholamines 1. Introduction Sclerotization of insect cuticle partly involves the reactions of functional groups of certain amino acid resi- dues of proteins in the presumptive exocuticle with qui- none metabolites of N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA) to form adducts and cross- links (Hopkins and Kramer, 1992; Andersen et al., 1996; Sugumaran, 1998). We have previously isolated and characterized several proteins from the tanning pupal cuticle of the tobacco hornworm, Manduca sexta, which contained covalently bonded NBAD. This was evi- denced by the release of N-β-alanylnorepinephrine * Corresponding author. Tel.: + 1-785-532-4722; fax: + 1-785-532- 6232. E-mail address: thopkins@oz.oznet.ksu.edu (T.L. Hopkins) 0965-1748/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII:S0965-1748(99)00091-0 (NBANE) and other catechols during acid hydrolysis of the purified proteins, and also by labeling of cuticular proteins in situ by 14 C-β-alanine (Okot-Kotber et al. 1994, 1996). These proteins had different N-terminal amino acid sequences and ranged in apparent molecular weight from 32 to 256 kD. Although they appeared to be unrelated in structure, they were similar in containing relatively large amounts of glycine, glutamic and aspartic acids, alanine and serine, which are typical of some other insect cuticular proteins. Small amounts of peptidyl DOPA were detected in some of the proteins, indicating the possible involvement of the catechol moi- ety in cross-linking of proteins (Okot-Kotber et al., 1994). The proteins also contained low to moderate amounts of histidine, an important amino acid in the pep- tide chain for covalent bonding to the N-acyldopamine quinones of NBAD and NADA (Schaefer et al., 1987; Christensen et al., 1991). Recently, we isolated and