title: Identification and molecular characterisation of a peritrophin gene, peritrophin-48, from the myiasis fly Chrysomya bezziana.
abstract: The peritrophic matrix lines the midgut of most insects and has important roles in digestion, protection of the midgut from mechanical damage and invasion by micro-organisms. Although a few intrinsic peritrophic matrix proteins have been characterised, no direct homologues of any of these proteins have been found in other insect species, even closely related species, suggesting that the peritrophic matrix proteins show considerable sequence divergence. We now report the identification of the cDNA and genomic DNA sequences of a Chrysomya bezziana homologue of the Lucilia cuprina intrinsic peritrophic matrix protein, peritrophin-48. The gene for C. bezziana peritrophin-48 spans 1315 bp and consists of three exons (65, 560 and 690 bp, respectively) separated by introns of 566 and 72 bp. The transcriptional start site, identified by a consensus of cDNA clones and primer extension analysis, is probably located 58 bp upstream from the start codon. However, there may be multiple start sites for transcription. Two potential TATA boxes and a consensus arthropod transcription initiator are located within 134 bp of sequence upstream of the putative transcriptional start site suggesting that this region contains the gene promoter. Immuno-fluorescence localization demonstrated that C. bezziana peritrophin-48 was localised to the larval peritrophic matrix. Protein fold recognition analysis indicated structural similarities between peritrophin-48 and wheatgerm lectin. As wheatgerm lectin binds chitin, this result suggested that C. bezziana peritrophin-48 may also bind chitin, a constituent of the peritrophic matrix. Chitin binding studies with a recombinant peritrophin-48 protein confirmed that it binds chitin. A Drosophila melanogaster homologue of peritrophin-48 encoded in an EST and a genomic sequence was also identified. The pairwise percentage identities of the deduced amino acid sequences for the peritrophin-48 homologues from the three higher Dipteran species were relatively low, ranging between 32 and 42%. Despite this sequence variability, the predicted structure of these proteins, dictated by five domains, each containing a characteristic distribution of six cysteines, was strictly conserved. It is concluded that considerable sequence variation can be tolerated in this protein because of the constraints imposed on the structure of the protein by an extensive disulphide bonded framework.
authors: CSIRO Livestock Industries, Molecular Animal Genetics Centre, Level 3, Gehrmann Laboratories, University of Queensland, Qld 4072, St Lucia, Australia. tony.vuocolo@tag.csiro.au
authors: T
authors: T
authors: Vuocolo
authors: C H
authors: CH
authors: Eisemann
authors: R D
authors: RD
authors: Pearson
authors: P
authors: P
authors: Willadsen
authors: R L
authors: RL
authors: Tellam
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D018076:DNA, Complementary; D004175:Diptera / Q000235:genetics; D004331:Drosophila melanogaster; D017344:Genes, Insect; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010843:Pichia; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D019476:Insect Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C434684:peritrophin-48 protein, Chrysomya bezziana