Structure-Function Analysis of the Anopheles gambiae LRIM1/APL1C Complex and its Interaction with Complement C3-Like Protein TEP1

Abstract

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.

Anopheles Midgut FREP1 Mediates Plasmodium Invasion

“…These interactions and the activity of chitinase, coupled with the digestive activity of plasmin on the ookinete surface (39), may result in a disruption of the PM structure, ultimately facilitating parasite invasion. After ookinetes cross the PM to invade midgut epithelial cells other proteins in mosquito hemolymph may perhaps interact with parasites and impact infection intensity (40,41). By extension, our model predicts that blocking FREP1 activity or interaction with Plasmodium parasites will significantly reduce the capacity for mosquitoes to transmit malaria.…”

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota

“…We show that the LRR proteins APL1A and APL1C play a role in midgut antiviral defense against virus infection. The role of these molecules as protective factors against Plasmodium infection is well documented (34,36,(53)(54)(55). APL1C is a subunit, along with LRIM1 and Tep1, in a ternary immune complex with activity against rodent malaria parasites (34,35,56), whereas its paralog APL1A, but not APL1C, protects against P. falciparum infection (36).…”

AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae

“…Expression of three TEP genes (TEP1, TEP3, and TEP12) was significantly up-regulated by wounding. TEP1 and TEP3 cooperate to TGase2-mediated Killing of P. falciparum in A. gambiae promote phagocytosis of Gram-positive and Gram-negative bacteria (50) and in antiparasitic responses (51,52). TEP1 circulates in the mosquito hemolymph as a trimeric complex comprising two leucine-rich repeat proteins, APL1C and LRIM1 (53,54).…”