A conserved domain in arthropod cuticular proteins binds chitin.

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).