Abstract
Malaria, the world’s most devastating parasitic disease, is transmitted between humans by mosquitoes of the Anopheles genus. An. gambiae is the principal malaria vector in Sub-Saharan Africa. The 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 CXCXC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 form higher oligomeric states at physiological pH. Both lectins bind specific sugars, including glycosaminoglycan motifs with β1-3/β1-4 linkages between glucose, galactose and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is found to function in vivo, reversing the enhancement of phenol oxidase 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.
Highlights
The innate immune response of An. gambiae to malaria parasites in an infectious blood meal is a significant factor influencing the prevalence and vectoral capacity of mosquitoes in a population[1,2]
An. gambiae has a complement-like immune response centered upon thioester-containing protein 1 (TEP1) that effectively targets Plasmodium ookinetes following their traversal of the midgut epithelium, prior to their transformation into oocysts[3,4,5,6,7]
In An. albimanus CTL4/CTLMA2 is antagonistic towards both P. berghei and P. falciparum[22]. This has led to the hypothesis that the functions of these two proteins or associated cofactors, have diverged and their mechanistic involvement in regulating infection intensity and melanization is unlinked. These data led us to address the question, what is the molecular structure and function of CTL4/CTLMA2 and to what extent is it conserved throughout the Anopheles genus? We report that CTL4/CTLMA2 intermolecular disulfide bond formation can occur via any two cysteines of the CXCXC motif, and that the two proteins can form higher-order oligomers via complementary electrostatic interactions
Summary
The innate immune response of An. gambiae to malaria parasites (genus Plasmodium) in an infectious blood meal is a significant factor influencing the prevalence and vectoral capacity of mosquitoes in a population[1,2]. An. gambiae has a complement-like immune response centered upon thioester-containing protein 1 (TEP1) that effectively targets Plasmodium ookinetes following their traversal of the midgut epithelium, prior to their transformation into oocysts[3,4,5,6,7]. Many proteins have a CTL fold but lack the Ca2+-binding site and do not bind sugars, i.e. they are CTLDs but not C-type CRDs14. Two types of lectins in the immune system are collectins and selectins Collectins such as mannose-binding protein (MBP) and surfactant protein A and D (SP-A, SP-D) are mannose-type secreted homo-oligomers that bind to pathogen surfaces and trigger innate immune responses such as complement. The CTL proteins CTL4 and CTLMA2 were first reported to influence the immune response of An. gambiae to P. berghei infection coincident with that of the first LRIM family member LRIM117. Knockdown of CTL4 (dsCTL4) or CTLMA2 (dsCTLMA2) results in significantly reduced numbers of oocysts and increased melanization, implying a susceptible or agonist phenotype
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