Insect Immune Activation by Apolipophorin III Is Correlated with the Lipid-Binding Properties of This Protein

Abstract

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N-->D) and/or 68 (K-->E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

Pre-exposure to yeast protects larvae of Galleria mellonella from a subsequent lethal infection by Candida albicans and is mediated by the increased expression of antimicrobial peptides

“…This protein has been shown to increase and stimulate the immune response and its ability to stimulate an immune response is associated with its lipid binding capabilities [13]. Here there was a 5-fold decrease in the precursor form of this protein in C. albicans challenged larvae whichwould suggest that the protein's active form was increased.…”

“…Mass spectrometry analysis of tryptic-digested proteins or naturally occurring peptides (peptidomics) has also been used to quantify the changes in protein expression and the induction of novel proteins following infection [13]. Recent studies have utilised two-dimensional (2D) analysis of Drosophila haemolymph and Anopheles gambiae [14] and many groups have reported data concerning protein expression and induction in Drosophila utilising a proteomic approach [15e17].…”

“…RNA was extracted from whole larvae over 48 h and cDNA was generated. The genes that were examined coded for galiomicin, a defensin identified in G. mellonella [13], a cysteine-rich antifungal peptide gallerimycin [25], transferrin, an iron binding protein and an inducible metalloproteinase inhibitor (IMPI) from G. mellonella [12] (Fig. 3).…”

A Novel Role for an Insect Apolipoprotein (Apolipophorin III) in β-1,3-Glucan Pattern Recognition and Cellular Encapsulation Reactions

“…Lipid-associated apoLp-III complexes were synthesized for functional assays, as several reports indicated that apoLp-III functions immunologically only when bound to lipid (15,(22)(23)(24). To produce the complex, 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) was incubated with apoLp-III at a ratio of 2:1, respectively, as described by Dettloff and Wiesner (15).…”

“…Furthermore, apoLp-III stimulates increases in hemolymph antibacterial activity (3) and superoxide production by blood cells (hemocytes) (15). Under normal conditions in larval hemolymph, apoLp-III exists abundantly in a folded, lipid-free state (21); however, recent studies (15,(22)(23)(24) strongly suggest that lipid-free apoLp-III must first convert to a lipid-associated conformation to function immunologically, as is the case for mammalian apoE (10). Until now, however, no role has been identified for apoLp-III in the binding of ␤-1,3-glucans released systemically from fungal cell walls.…”