Bok L. Lee

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

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and β-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of β-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

Crystal structure of a clip‐domain serine protease and functional roles of the clip domains

Clip‐domain serine proteases (SPs) are the essential components of extracellular signaling cascades in various biological processes, especially in embryonic development and the innate immune responses of invertebrates. They consist of a chymotrypsin‐like SP domain and one or two clip domains at the N‐terminus. Prophenoloxidase‐activating factor (PPAF)‐II, which belongs to the noncatalytic clip‐domain SP family, is indispensable for the generation of the active phenoloxidase leading to melanization, a major defense mechanism of insects. Here, the crystal structure of PPAF‐II reveals that the clip domain adopts a novel fold containing a central cleft, which is distinct from the structures of defensins with a similar arrangement of cysteine residues. Ensuing studies demonstrated that PPAF‐II forms a homo‐oligomer upon cleavage by the upstream protease and that the clip domain of PPAF‐II functions as a module for binding phenoloxidase through the central cleft, while the clip domain of a catalytically active easter‐type SP plays an essential role in the rapid activation of its protease domain.

Crystal Structure of the Serine Protease Domain of Prophenoloxidase Activating Factor-I

A family of serine proteases (SPs) mediates the proteolytic cascades of embryonic development and immune response in invertebrates. These proteases, called easter-type SPs, consist of clip and chymotrypsin-like SP domains. The SP domain of easter-type proteases differs from those of typical SPs in its primary structure. Herein, we report the first crystal structure of the SP domain of easter-type proteases, presented as that of prophenoloxidase activating factor (PPAF)-I in zymogen form. This structure reveals several important structural features including a bound calcium ion, an additional loop with a unique disulfide linkage, a canyon-like deep active site, and an exposed activation loop. We subsequently show the role of the bound calcium and the proteolytic susceptibility of the activation loop, which occurs in a clip domain-independent manner. Based on biochemical study in the presence of heparin, we suggest that PPAF-III, highly homologous to PPAF-I, contains a surface patch that is responsible for enhancing the catalytic activity through interaction with a nonsubstrate region of a target protein. These results provide insights into an activation mechanism of easter-type proteases in proteolytic cascades, in comparison with the well studied blood coagulation enzymes in mammals.

Auxiliary role for d-alanylated wall teichoic acid in Toll-like receptor 2-mediated survival of Staphylococcus aureus in macrophages

We previously reported that Staphylococcus aureus avoids killing within macrophages by exploiting the action of Toll‐like receptor 2 (TLR2), which leads to the c‐Jun N‐terminal kinase (JNK)‐mediated inhibition of superoxide production. To search for bacterial components responsible for this event, a series of S. aureus mutants, in which the synthesis of the cell wall was interrupted, were screened for the level of JNK activation in macrophages. In addition to a mutant lacking the lipoproteins that have been suggested to act as a TLR2 ligand, two mutant strains were found to activate the phosphorylation of JNK to a lesser extent than the parental strain, and this defect was recovered by acquisition of the corresponding wild‐type genes. Macrophages that had phagocytosed the mutant strains produced more superoxide than those engulfing the parental strain, and the mutant bacteria were more efficiently killed in macrophages than the parent. The genes mutated, dltA and tagO, encoded proteins involved in the synthesis of d‐alanylated wall teichoic acid. Unlike a cell wall fraction rich in lipoproteins, d‐alanine‐bound wall teichoic acid purified from the parent strain by itself did not activate JNK phosphorylation in macrophages. These results suggest that the d‐alanylated wall teichoic acid of S. aureus modulates the cell wall milieu for lipoproteins so that they effectively serve as a ligand for TLR2.

Structural characteristics of tenecin 3, an insect antifungal protein

Tenecin 3, an antifungal protein, previously isolated from the insect Tenebrio molitor, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 remain unknown. As an initial step to study the mechanism and functions, physical and structural properties of tenecin 3 were examined by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy. These analyses suggest that tenecin 3 has a propensity of random structure with very loose turn‐like elements. The CD results also indicate that this random structural propensity is not significantly affected by temperature, pH, and by the presence of organic solvents or sodium dodecyl sulfate (SDS) micelles. However, the hydrodynamic studies suggest that tenecin 3 is not in extended form in spite of its random structural feature.

Preliminary X-ray crystallographic analysis of the catalytic domain of prophenoloxidase activating factor-I

Clip-domain serine proteases (SPs) have been identified in invertebrates as crucial enzymes that are involved in diverse extracellular signalling pathways. Prophenoloxidase (proPO) activating factor-I (PPAF-I), a catalytically active clip-domain SP, cleaves proPO. To date, no crystal structures of a catalytically active clip-domain SP have been determined. Here, the results of crystallization and preliminary X-ray analysis of the SP domain of PPAF-I are reported. The crystal of the PPAF-I SP domain was obtained using the hanging-drop vapour-diffusion method in a precipitant solution containing 0.15 M lithium sulfate, 30% polyethylene glycol 4000 and 0.1 M Tris-HCl pH 8.0. The crystal diffracts X-rays to 1.7 angstroms resolution using a synchrotron-radiation source. The crystal belongs to space group P2(1)2(1)2(1), with one molecule in the asymmetric unit and unit-cell parameters a = 38.3, b = 53.3, c = 116.6 angstroms, alpha = beta = gamma = 90 degrees. A molecular-replacement solution has been found using kallikrein as a starting model, resulting in an interpretable electron-density map.