Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana

The peritrophic matrix lines the midgut of most insects and has important r oles in digestion, protection of the midgut from mechanical damage and inva sion by micro-organisms. Although a few intrinsic peritrophic matrix protei ns have been characterised, no direct homologues of any of these proteins h ave been found in other insect species, even closely related species, sugge sting that the peritrophic matrix proteins show considerable sequence diver gence. We now report the identification of the cDNA and genomic DNA sequenc es of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic perit rophic matrix protein. peritrophin-48. The gene for C. bezziana peritrophin -48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respecti vely) separated by introns of 566 and 72 bp. The transcriptional start site , identified by a consensus of cDNA clones and primer extension analysis, i s probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a c onsensus arthropod transcription initiator are located within 134 bp of seq uence upstream of the putative transcriptional start site suggesting that t his region contains the gene promoter. Immune-fluorescence localization dem onstrated that C. bezziana peritrophin-48 was localised to the larval perit rophic matrix. Protein fold recognition analysis indicated structural simil arities between peritrophin-48 and wheatgerm lectin, As wheatgerm lectin bi nds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studie s with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST an d a genomic sequence was also identified. The pairwise percentage identitie s of the deduced amino acid sequences for the peritrophin-48 homologues fro m the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of the se proteins, dictated by five domains, each containing a characteristic dis tribution of six cysteines, was strictly conserved. It is concluded that co nsiderable sequence variation can be tolerated in this protein because of t he constraints imposed on the structure of the protein by an extensive disu lphide bonded framework. (C) 2001 Elsevier Science Ltd. All rights reserved .