Anopheles gambiae TEP1 forms a complex with the coiled-coil domain of LRIM1/APL1C following a conformational change in the thioester domain

Abstract

The complement-like protein thioester-containing protein 1 (TEP1) is a key factor in the immune response of the malaria vector Anopheles gambiae to pathogens. Multiple allelic variants of TEP1 have been identified in laboratory strains and in the field, and are correlated with distinct immunophenotypes. TEP1 is tightly regulated by conformational changes induced by cleavage in a protease-sensitive region. Cleaved TEP1 forms a soluble complex with a heterodimer of two leucine-rich repeat proteins, LRIM1 and APL1C, and precipitates in the absence of this complex. The molecular structure and oligomeric state of the TEP1/LRIM1/APL1C complex is unclear. We have analyzed the stability of the cleaved form of four TEP1 alleles. Soluble TEP1 forms exhibit significant variation in stability from hours to days at room temperature. Stability is correlated with allelic variation within two specific loops in direct proximity to the thioester bond. The variable loops are part of an interface between the TED and MG8 domains TEP1 that protect the thioester from hydrolysis. Engineering specific disulfide bonds to prevent separation of the TED-MG8 interface stabilizes the cleaved form of TEP1 for months at room temperature. The C-terminal coiled-coil domain of the LRIM1/APL1C complex is sufficient to stabilize the cleaved form of TEP1 in solution but cleaved forms of disulfide-stabilized TEP1 do not interact with LRIM1/APL1C. This implies that formation of the TEP1cut/LRIM1/APL1C complex is dependent on the same conformational change that induces the precipitation of cleaved TEP1. Author SummaryThe mosquito Anopheles gambiae is the principal vector for malaria in Sub-Saharan Africa. A mosquitos own immune system affects how readily it transmits disease. A protein in A. gambiae called TEP1 is responsible for targeting malaria parasites that traverse the mosquitos midgut. TEP1 has multiple alleles and some are associated with a stronger immune response to malaria than others. How genetic variability in TEP1 is linked to phenotypic diversity is not understood. We show that the variation between TEP1 alleles affects the stability of the protein in solution. We also show that the different TEP1 alleles have a wide range in stability of the protein, from hours to days. Engineering disulfide bonds into TEP1 can increase this stability to months. TEP1 activity in vivo is maintained by a complex of two leucine-rich proteins called LRIM1 and APL1C, which binds TEP1 through its C-terminal coiled-coil domain. We found that LRIM1/APL1C does not bind disulfide-stabilized TEP1, suggesting that LRIM1/APL1C binds to activated TEP1. This research advances our molecular understanding of a key immune response that affects the capacity of A. gambiae mosquitoes to transmit malaria.