THE ROLE OF DITYROSINE FORMATION IN THE CROSS-LINKING OF CUT-2, THE PRODUCT OF A 2ND CUTICLIN GENE OF CAENORHABDITIS-ELEGANS

A second cuticlin gene, cut-2, of the nematode Caenorhabditis elegans, has been isolated and its genomic and cDNA sequences determined. The gene codes for a component of cuticlin, the insoluble residue of nemat ode cuticles. Conceptual translation of cut-2 reveals a 231-amino acid secreted protein which, like CUT-I, begins with a putative signal pep tide of 16 residues. The central part of the protein consists of 13 re petitions of a short hydrophobic motif, which is often degenerated wit h substitutions and deletions. Parts of this motif are present also in CUT-1 (Caenorhabditis elegans) as well as in several protein componen ts of the larval cuticle and of the eggshell layers of various insects (Locusta migratoria, Ceratitis capitata and Drosophila species). Thes e sequence similarities are related to the similar functions of these proteins: they are all components of extracellular insoluble protectiv e layers. Immunolocalisation and transcription analysis suggest that C UT-2 contributes to the cuticles of all larval stages and that it is n ot stage-specific. Analysis by reverse transcriptase-PCR suggests that transcription is not continuous throughout larval development but occ urs in peaks which precede the moults. Dityrosine has been detected in the cuticle of nematodes and of insects; formation of dityrosine brid ges may be one of the cross-linking mechanisms contributing to the ins olubility of cuticlins. Recombinant, soluble CUT-2 is shown to be an e xcellent substrate for an in vitro cross-linking reaction, catalysed b y horseradish peroxidase in the presence of H2O2, which results in the formation of insoluble, high-molecular weight CUT-2 and of dityrosine .