Contet, A. | bioRxiv Science

bioRxiv2019

C-type lectins CTL4 and CTLMA2: conserved heterodimeric structure and glycan specificity in Anopheles mosquitoes

AbstractThe C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising {beta}1-3/{beta}1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection. Author SummaryMosquitoes of the genus Anopheles are vectors for the single-celled parasite Plasmodium, the causative agent of malaria. Mosquitoes, like all insects, utilize the process of melanization for both wound healing and defense against pathogens. CTL4 and CTLMA2 are two proteins found in Anopheles mosquitoes that act as inhibitors of melanization, so understanding their molecular function is important to understanding the immune response of Anopheles mosquitoes to Plasmodium infection. We have purified CTL4 and CTLMA2 from two species of Anopheles and studied their molecular properties with a variety of biochemical and biophysical techniques. We also verified that our purified protein is functional by injecting it into mosquitoes. We learned that CTL4 and CTLMA2 are joined together by a disulfide bond between any one of three cysteine residues near the N-terminus of each protein. The CTL4/CTLMA2 complex is compact, but can associate into larger structures in solution, probably because of a loop in each protein that carries an opposite charge. The proteins cooperatively bind calcium and sugars, specifically glycosaminoglycan sugars, which are typically present in the connective tissues of insects. This information will aid in further investigations of the function of CTL4 and CTLMA2.

biochemistry↗

bioRxiv2019

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

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.

immunology↗