Ji-Won Park

Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects

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 lysozyme-mediated 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.

A Three-step Proteolytic Cascade Mediates the Activation of the Peptidoglycan-induced Toll Pathway in an Insect

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 pro-protein 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.

Proteolytic cascade for the activation of the insect Toll pathway induced by the fungal cell wall component

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation.

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and β-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

Predictors for lymph node metastasis in T1 colorectal cancer.

BACKGROUND AND STUDY AIMSnIt is critical that the risk of lymph node metastasis (LNM) is evaluated for determining the suitability of endoscopic resection for T1 colorectal cancer (CRC). Reported risk factors for LNM in completely resected T1 CRC are deep submucosal invasion, grade 3, angiolymphatic invasion, and budding. The aim of the present study was to identify the histopathologic factors associated with LNM in T1 CRC.nnnPATIENTS AND METHODSnThe study involved 435 patients with T1 CRC treated by endoscopic or surgical resection between January 2001 and April 2010 at the National Cancer Center, Korea. The 435 patients were classified into two groups - those undergoing surgical resection (n = 324) and those undergoing endoscopic resection (n = 111). In the surgically resected group, details regarding depth of submucosal invasion, angiolymphatic invasion, tumor grade, budding, and background adenoma (BGA) were evaluated with respect to presence or absence of LNM. In the endoscopically resected group, the results of follow-ups and additional salvage surgeries were studied.nnnRESULTSnIn the surgically resected group, LNM was detected in 42 patients (13.0 %). Grade 3, angiolymphatic invasion, budding, and the absence of BGA were identified as factors associated with LNM in univariate and multivariate analyses (P < 0.05). Among the 50 patients in the endoscopically resected group with high risk, three were diagnosed as being LNM-positive during the follow-up period. There was no LNM in the endoscopically resected group with low risk.nnnCONCLUSIONSnGrade 3, angiolymphatic invasion, budding, and the absence of BGA are the risk factors that predict LNM in patients with T1 CRC. In cases where endoscopically resected T1 CRC has no risk factor, cautious follow-up could be recommended. However, if the tumor has any risk factor, additional surgical resection should be considered.

Clinicopathological differences of laterally spreading tumors of the colorectum according to gross appearance

BACKGROUND AND STUDY AIMSnLaterally spreading tumors (LST) are classified into two subtypes, with the nongranular type harboring a higher risk of (pre)malignant changes than the granular type. Further subdifferentiation into two subgroups each has been suggested, but the clinical significance of such a subdifferentiation has not previously been studied in detail in larger numbers.nnnPATIENTS AND METHODSnOut of 6499 patients diagnosed with colorectal adenomas between January 2006 and November 2008, 153 patients (2.35 %) had 158 LSTs, 96 with a granular and 62 with a nongranular pattern. The former group was subdivided into homogeneous and nodular mixed, the latter group into flat elevated and pseudodepressed. Clinical and histopathological parameters were compared among the four subtypes.nnnRESULTSnParameters were variably distributed between the four groups, with nodular mixed tumors being larger than the other three types ( P < 0.0001). As in other studies, malignant transformation and premalignant lesion (HGIN/CIS) were more frequent in nodular mixed than in homogeneous tumors (45.0 % vs. 5.6 %, P < 0.001), and also more common in pseudodepressed than in flat elevated tumors (41.7 % vs. 13.2 %, P = 0.011). Submucosal invasive cancer was present in 8.3 % of nodular mixed tumors, 7.9 % of flat elevated, and 12.5 % of pseudodepressed, while it was absent in homogeneous tumors. Serrated adenoma was identified in 10.8 % of all LSTs, and sessile serrated adenoma tended to be more common in flat elevated tumors.nnnCONCLUSIONSnFurther subdifferentiation of the LST lesions to identify lesions at risk of malignant transformation makes most sense in the granular type. Among nongranular LSTs, both subtypes carry a significant risk.

Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects.

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.

Comparative study of NOTES rectosigmoidectomy in a swine model: E-NOTES vs. P-NOTES.

BACKGROUND AND STUDY AIMSnSeveral reports have demonstrated the safety of pure natural-orifice transluminal endoscopic surgery (P-NOTES) using transanal endoscopic microsurgery (TEM) and embryonic NOTES (E-NOTES; laparoscopic surgery through the umbilicus). This study was performed to compare the safety and applicability of NOTES rectosigmoidectomy between E-NOTES and P-NOTES in a swine model.nnnPATIENTS AND METHODSnE-NOTES was conducted through a single port using laparoscopic instruments (n = 11). P-NOTES was performed using TEM with transgastric endoscopic assistance (n = 11). Gastrotomies were created using a needle knife and the balloon dilatation technique, and closed using T-anchors. Blood samples were collected to evaluate changes in systemic cytokine levels during the preoperative and postoperative periods; operative outcomes were also evaluated and compared between the groups. The necropsy findings were recorded after sacrifice at 1 week after the procedure.nnnRESULTSnThe mean operative time for P-NOTES was significantly longer than that for E-NOTES (239 vs. 103 minutes, P < 0.001). The mean distance from the anal verge to colorectal anastomosis in the P-NOTES group was significantly less than that in the E-NOTES group (2.9 vs. 17.6 cm, P < 0.001). On necropsy, the complication rate of P-NOTES was higher than that of E-NOTES, but without statistical significance (54.5 % vs. 18.2 %, P = 0.091). The differences in changes in TNF-α, C-reactive protein, interleukin-6, and interleukin-1β between P-NOTES and E-NOTES were not significant.nnnCONCLUSIONSnE-NOTES rectosigmoidectomy in the swine model is safe, but remains challenging for use in pelvic dissection. P-NOTES rectosigmoidectomy using TEM may be a promising tool for pelvic dissection, but the transgastric approach involves a high degree of risk.