Solution structure, glycan specificity and of phenol oxidase inhibitory activity of Anopheles C-type lectins CTL4 and CTLMA2.

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 ß1-3/ß1-4 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.

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 exhibit significant variation in stability from hours to days at room temperature. In particular, the refractory allele TEP1*R1 is significantly more stable than the susceptible allele TEP1*S1. This raises the question of whether the stability of cleaved TEP1 is linked to allelic variation and varying immunophenotypes. We have analyzed the stability of the cleaved form of additional TEP1 alleles and constructs. We show that 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 of 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. 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. 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 related to the conformational change that induces the precipitation of cleaved TEP1.

[Immunotoxins and immunocytokines]. / Immunotoxines et immunocytokines.

Cytokines and biological toxins represent two potent classes of biomolecules that have long been explored for their potential as therapeutics. Considerable side effects and poor pharmacokinetics frequently observed with both have limited their broad application. Recombinant protein engineering has allowed the construction of immunocytokines and immunotoxins that seek to exploit the advantageous properties of immunoglobulins to address these issues. Whole antibodies, antibody fragments, constant domains and derivatives have been fused genetically to a range of cytokines and toxins. This review considers the strategies that have been employed and the problems sought to be resolved in the clinical evaluation of this class of biotherapeutic.

TITLE: Immunotoxines et immunocytokines. ABSTRACT: Les cytokines et les toxines biologiques représentent deux classes de biomolécules qui ont longtemps été explorées pour leur potentiel thérapeutique. Des effets secondaires considérables et des mauvaises propriétés pharmacocinétiques sont fréquemment observés chez chacune d'elles, ce qui limite leur application. L'ingénierie des protéines recombinantes a permis la création d'immunocytokines et d'immunotoxines qui visent à utiliser les propriétés avantageuses des immunoglobulines, pour résoudre ces problèmes. Des anticorps entiers, des fragments d'anticorps, des domaines constants et des dérivés ont été génétiquement fusionnés à une gamme de cytokines et de toxines. Cette revue présente les stratégies déployées et les problèmes à résoudre au cours de l'évaluation clinique pour cette classe de biothérapeutiques.

Complement-Related Regulates Autophagy in Neighboring Cells.

Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. Here, we report that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program.

Arthropod Innate Immune Systems and Vector-Borne Diseases.

Arthropods, especially ticks and mosquitoes, are the vectors for a number of parasitic and viral human diseases, including malaria, sleeping sickness, Dengue, and Zika, yet arthropods show tremendous individual variation in their capacity to transmit disease. A key factor in this capacity is the group of genetically encoded immune factors that counteract infection by the pathogen. Arthropod-specific pattern recognition receptors and protease cascades detect and respond to infection. Proteins such as antimicrobial peptides, thioester-containing proteins, and transglutaminases effect responses such as lysis, phagocytosis, melanization, and agglutination. Effector responses are initiated by damage signals such as reactive oxygen species signaling from epithelial cells and recognized by cell surface receptors on hemocytes. Antiviral immunity is primarily mediated by siRNA pathways but coupled with interferon-like signaling, antimicrobial peptides, and thioester-containing proteins. Molecular mechanisms of immunity are closely linked to related traits of longevity and fertility, and arthropods have the capacity for innate immunological memory. Advances in understanding vector immunity can be leveraged to develop novel control strategies for reducing the rate of transmission of both ancient and emerging threats to global health.

Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening.

Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.

Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active.

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis-Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.