title: A conserved domain in arthropod cuticular proteins binds chitin.
abstract: Many insect cuticular proteins include a 35-36 amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin. Provocatively, it has no sequence similarity to the well-known cysteine-containing chitin-binding domain found in chitinases and some peritrophic membrane proteins. Using fusion proteins expressed in E. coli, we show that an extended form of the R&R consensus from proteins of hard cuticles is necessary and sufficient for chitin binding. Recombinant AGCP2b, a putative cuticular protein from the mosquito Anopheles gambiae, was expressed in E. coli and the purified protein shown to bind to chitin beads. A stretch of 65 amino acids from AGCP2b, including the R&R consensus, conferred chitin binding to glutathione-S-transferase (GST). Directed mutagenesis of some conserved amino acids within this extended R&R consensus from hard cuticle eliminated chitin binding. Thus arthropods have two distinct classes of chitin binding proteins, those with the chitin-binding domain found in lectins, chitinases and peritrophic membranes (cysCBD) and those with the cuticular protein chitin-binding domain (non-cysCBD).
authors.affiliation: Department of Biology, Northern Michigan University, Marquette, MI 49855, USA. jrebers@nmu.edu
authors.forename: J E
authors.initials: JE
authors.lastname: Rebers
authors.forename: J H
authors.initials: JH
authors.lastname: Willis
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles; D001181:Arthropods; D002482:Cellulose / Q000378:metabolism; D002686:Chitin / Q000378:metabolism*; D016384:Consensus Sequence; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; C047663:cuticle proteins, insects; D002686:Chitin; D002482:Cellulose