A molting protein from Calpodes ethlius (Lepidoptera, Hesperiidae) and its relation to the structure of insect cuticle.

摘要

Molting in insects requires the degradation of the cuticle by enzymes in the molting fluid. Protease and chitinase activities have been described in insect molting fluid, but nothing is known about the protein-chitin bonds in cuticle and whether enzymes are needed to cleave these bonds at molting. This work describes the isolation and characterization of a new molt-associated protein that may degrade protein-chitin bonds in cuticle. CECP22 (Calpodes ethlius Cuticular Protein 22 kDa) was purified from prepupal cuticles of 5{dollar}sp{lcub}rm th{rcub}{dollar} instar Calpodes caterpillars. The corresponding cDNA was isolated by antibody screening of an epidermal expression library. The mRNA is expressed in the epidermis and fat body during the intermolt. The protein is secreted in the intermolt hemolymph, where it accumulates as an inactive enzyme precursor. At molting, the hemolymph store provides protein for both internal and surface integument. The protein is post-translationally processed in the cuticle by the cleavage and amidation of the C-terminal end, suggesting a mechanism for enzyme activation.; The amino acid sequence of CECP22 shares some similarity with the sequence of bacteriophage T7 N-acetylmuramoyl-L-alanine amidase, which has as substrate an amidic bond in the bacterial cell wall. The substrate of CECP22 could be an amidic bond in the insect cuticle, involving amino acid inkers between proteins and chitin. Based on their occurence in the cuticle, their association with chitin fibers and their release at the time of CECP22 cleavage, candidate linkers are aspartic acid, glutamic acid, serine, threonine and histidine.; Besides using amino acids as linkers for stabilizing of chitin chains and binding proteins, cuticle stores most of the amino acids in a water soluble pool during intermolt.; The timing of CECP22 expression in development, the features of its amino acid sequence, the site and correlation of its cleavage with the release of amino acids at molting, all suggest that the protein may have a role in cleaving chitin-peptide bonds. This is a new type of molting enzyme, that could be a prerequisite for digestion of the cuticle by chitinases and proteases.