The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle proprotein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozymemediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.innate immunity ͉ pattern ͉ prophenoloxidase ͉ Toll

Summary Coupling between certain pathogen‐associated molecular patterns and corresponding pattern recognition receptors of endothelial cells is important for the mediation of vascular inflammatory responses. Mannose‐binding lectin (MBL) recognizes certain carbohydrate structures of microbes and subsequently activates the complement system as well as facilitates the phagocytosis of targets. We investigated whether MBL can intervene in the interaction between bacterial lipopolysaccharide (LPS) and endothelial cells to modulate subsequent inflammatory responses. In response to LPS, human umbilical vein endothelial cells (HUVEC) produced various cytokines/chemokines. Addition of the purified human MBL/MBL‐associated serine proteases (MASP) complex or recombinant human MBL enhanced LPS‐mediated cytokine/chemokine secretion by HUVEC, including interleukin‐8 (IL‐8), IL‐6 and monocyte chemoattractant protein‐1 in a dose‐dependent manner. This enhancing effect was ameliorated by the addition of anti‐MBL antibody or mannan. Among the cytokines/chemokines we analysed, IL‐6 showed the greatest increase of secretion in the presence of native MBL/MASP complex or recombinant MBL. MBL, regardless of its association with MASP, alters LPS‐mediated cytokine/chemokine secretion of HUVEC. Besides the well‐known functions of MBL, to activate the lectin–complement pathway and to facilitate clearance of targets, alteration of cytokine/chemokine secretion may provide an additional role for MBL in modulating vascular inflammation.

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Tool-origin compensation is important in high-precision micro-machining, as the tool-origin can become changed by the tool replacement. It is desirable to use a simple system, preferably consisting of a single light source and a single detector. To date, such systems have been restricted to measuring the tool position in two dimensions. We describe a measurement system that uses a single laser diode and a single photodetector that can measure change in the tool displacement in three dimensions. The sensor apparatus uses the principle of occlusion to measure the tool location, based on a modified version of Shin's measurement models. The resolution and repeatability were evaluated and the tool-origin compensation ability of the optical sensor was demonstrated by characterizing the machining error.