The extended loop of the C-terminal carbohydrate-recognition domain of Manduca sexta immunlectin-2 is important for ligand binding and functions

Our previous research showed that immunlectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and ƒÒ-1,3-glucan, and IML-2 plays an important role in cellualr encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis, and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2ƒ´220-244 and IML-2ƒ´220-257), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2ƒ´220-244 and IML-2ƒ´220-257, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions.