Insect Bioehem. Vol. 16, No. 3, pp. 441~,47. 1986 0020-1790/86 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Press Ltd CHARACTERIZATION OF CUTICULAR PROTEINS FROM THE MIGRATORY LOCUST, LOCUSTA MIGRATORIA SVEND OLAV ANDERSEN, 1 PETER HOJRUP 2 and PETER ROEPSTORFF 2 qnstitute of Biological Chemistry A, Copenhagen University, Universitetsparken 13, DK 2100 Copenhagen and 2Department of Molecular Biology, Odense University, Campusvej 55, DK 5230 Odense, Denmark (Received 17 June 1985; revised and accepted 21 August 1985) Abstract--Proteins were extracted from the still unhardened (teneral) cuticle of the migratory locust, Locusta migratoria. The proteins are soluble only at extreme pH-values and at low ionic strength, the solubility increases with decreasing temperature. The unhardened cuticle contains approx. 100 different proteins according to two-dimensional polyacrylamide gel electrophoresis. The majority of the proteins are very basic. The basicity and solubility properties of the proteins have necessitated development of modified electrophoretic procedures. The amino acid composition of the bulk protein shows that alanine, proline, glycine, valine and tyrosine constitute two thirds of the total amino acid content and that cysteine, methionine and tryptophan are absent. The proteins have been extracted from various parts of the cuticle and analysed by two-dimensional electrophoresis. Characteristic protein compositions were found for cuticle from the different body parts. Amino acid analyses of these extracts are strikingly similar. The only significant difference is in the glycine-alanine ratio. Cuticles that are destined to become hard are extremely rich in alanine, whereas the flexible parts of the cuticle are enriched in glycine. The results indicate that the proteins of locust cuticle constitute a group of structural proteins different from other known structural proteins. Key Word Index: Locusta migratoria, cuticle, protein mapping, amino acid composition, hydro- phobicity, solubility INTRODUCTION Insect cuticle is an extracellular layer, consisting mainly of proteins and chitin, which surrounds the whole animal (Neville, 1975; Hepburn, 1976). The cuticle functions both as a skeleton and as a barrier towards the environment, and it is also responsible for the coloration of most insects, either by pigments in the cuticle or by submicroscopic structures giving rise to physical colours. The ultrastructural or- ganization of cuticle follows a general plan in all insect groups: long filaments of chitin are embedded in a matrix of proteins, and most differences between cuticles are differences in the proteinaceous matrices and the way they are modified during maturation. Cuticles vary considerably in physical properties; most larval cuticles are very soft and flexible, and in the living animal they are kept distended by the internal pressure in the larvae, whereas large parts of the cuticle of adult insects are quite hard and inex- tensible, and specialized parts, such as mandibles, can be extremely hard. Some of these differences are due to differences in the degree of chemical stabilization (sclerotization) of the cuticular proteins (Andersen, 1974). The proteins are synthesized in the epidermal cells and secreted from the apical part of the cells. Some time after secretion, either before or after the insect has ecdyzed to the adult stage, the proteins can be sclerotized by an enzyme-catalyzed, oxidative in- corporation of phenolic material into the cuticle, presumably as cross-links between the protein mole- cules (Andersen, 1979). Sclerotization transforms the proteins from a soft, soluble material which is easily digested by proteolytic enzymes into a stiff, insoluble mass, which is much more resistant towards en- zymatic proteolysis. Various types of cuticle differ significantly in amino acid composition, indicating the presence of different proteins. As a rule, soft cuticles are characterized by a high content of polar amino acid residues, and hard cuticle tends to be rich in hydrophobic amino acids (Andersen, 1979). The old idea of a single, general cuticular protein, arthropodin, has long been aban- doned, and more and more evidence shows that all types of cuticle contain a large number of proteins (Willis et al., 1981). The exact pattern of proteins in a cuticle will presumably be of importance for its properties, but since only a few samples have been investigated it is too early to draw many conclusions. Proteins from larval cuticle of fruitflies, Drosophila melanogaster (Fristrom et al., 1978), larval cuticle of fleshflies, Sarcophaga bullata (Lipke et al., 1981), and cuticle from larvae, pupae and adults of the American silkmoth, Hyalophora cecropia (Willis et al., 1981), have been isolated and partly characterized. The total amino acid sequence has been obtained for several of the Drosophila larval cuticular proteins (Snyder et al., 1982); the published information for the other purified proteins concerns mainly molecular weights, isoelectric points and amino acid compositions. All these proteins resemble each other in having rela- tively low isoelectric points (pIs from 4 to 6), low molecular weights (8-25 kdal), and in being rich in polar amino acid residues. Only the easily extractable proteins have been characterized, and they account for about half of the 441 [ B 16'3 -A