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

Abstract

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 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 β1-3/β1-4 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 Summary Mosquitoes 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.