Symposium 3 : Innate Immnunity and Immune Regulation ; Role of Lectins involved in the innate immunity of arthropods

( Shun Ichiro Kawabata ) 한국생화학분자생물학회 (구 한국생화학회) 2000 추계학술대회집 Vol.2000 No.-

Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?

Liu, Haipeng,Wu, Chenglin,Matsuda, Yasuyuki,Kawabata, Shun-ichiro,Lee, Bok Luel,Sö,derhä,ll, Kenneth,Sö,derhä,ll, Irene Elsevier 2011 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol.35 No.1

<P><B>Abstract</B></P><P>Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, <I>Pl</I>-SPH2, was found and isolated as a 30kDa protein from hemocytes of the freshwater crayfish, <I>Pacifastacus leniusculus</I>, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type peptidoglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the <I>Pl</I>-SPH1 and lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of <I>Pl</I>-SPH2, <I>Pl</I>-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two <I>Pl</I>-SPHs are involved in this activation possibly by forming a complex with LGBP and without a PGRP.</P>

A Sapecin Homologue of Holotrichia diomphalia : Purification, Sepuencing and Determination of Disulfide Pairs

LEE, So Young,MOON, Hyun Joo,KAWABATA, Shun-ichiro,KURATA, Shoichiro,NATORI, Shunji,LEE, Bok Luel 부산대학교 유전공학연구소 1995 분자생물학 연구보 Vol.11 No.-

We purified and characterized a sapecin homologue, named holotricin 1, from the hemolymph of immunized larvae of a coleopteran insect, Holotrichia. We determined its complete amino acid sequence and three disulfide pairs. Holotricin 1 consisted of 43 amino acid residues and showed potent antibacterial activity against gram-positive bacteria, but antivacterial activity against gram-negative bacteria was not obvious.

Purification and Characterization of Prophenoloxidase from the Hemolymph of Coleopteran Insect, Holotrichia diomphalia Larvae

Kwon, Tae Hyuk,Lee, So Young,Lee, Ji Hye,Choi, Jae Sue,Kawabata, Shun-ichiro,Iwanaga, Sadaaki,Lee, Bok Luel 부산대학교 유전공학연구소 1997 분자생물학 연구보 Vol.13 No.-

Prophenoloxidase(pro-PO), a precursor of phenol oxidase(PO), was purified from the hemolymph of coleoptrean Holotrichia diomphalia larvag. The enzyme was purified to apparent homogeneity, in six steps of chromatorgraphy, using Sephadex G-100, CM-52, Dextran-sulfate Sepharose CL-6B, Phenly Sepharose CL-4B, Sephacryl S-200, and a Mono-Q column. The preparation exhibited a single band on SDS-PAGE. The proenzyme had a molecular weight of 158 kDa, as estimated by gel filtration. On SDS-PAGE under reducting. On the other hand, the purified pro-PO gave two well-separated peaks, named pro-PO-1 and pro-PO-2, on reverse-phase HPLC. Amino acid compositions of both proteins were indistinguishable, thereby suggesting the presence of an allelic variant or an isoprotein. On dextran-sulfate Sepharose CL-6B chromatorgraphy, a fraction containing pro-phenoloxidase activating enzyme(S)(PPAE fraction), free from pro-PO, was also separated. In reconstitution experimentsm, the astivation of purified pro-PO by PPAE fraction was observed in the presence of 5 mM Ca^2+, with a specific limited proteolysis. The NH_2-terminal sequence of generated PO was determined to be NH_2-Phe-Gly-Glu-Asp-Asp-. The activated PO oxidized o-diphenols but did not oxidize mono-phenol and p-diphenol substrates. The purified pro-PO was not activated by trypsin, α-chymotrypsin, and SDS.

In vitro activation of pro-phenol-oxidase by two kinds of pro-phenol-oxidase-activating factros isolated from hemolymph of coleopteran, Holotrichia diomphalia larvae

LEE, So Young,KWON, Tae Hyuk,HYUN, Ji Hoon,CHOI, Jae Sue,KAWABATA, Shun-Ichiro,IWANAGA Sadaaki,LEE, Bok Luel 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

Previously, we purtified and characterized a pro-phenol oxidase(pro-Po) of 79 kDa from coleopteran inset, Holotrichia diomphalia larvae[Kwon et al.(1997) Mol.Cells 7,90-97]. Here, we describe the indentification of two pro-PO activation factors(PPAF), named PPAF-Ⅰand PPAF-Ⅱ, directly involved in the activation of the isolated pro-PO. When pao-PO was incubated with either PPAF-Ⅰ or PPAF-Ⅱ, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-Ⅰand PPAF-Ⅱ specifically exhibited phenol oxidase activity. The purified PPAF-Ⅰ with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity aganist fluorogenic peptide substrate, tert-butoxycarbonylphenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02mM p-nitrophenyl-p'-guanidinobenzoate·HCl and diisopropylflurophosphate. the NH_2-treminal sequence of PPAF-Ⅰ had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-Ⅱ had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel fitration, indicating an oligomeric protein. The NH_2-terminal sequence of PPAF-Ⅱ showed no similarity to known proteins.PPAF-Ⅱ exhibited no amidase activity aganist the fluorogenic substrates. Reconstitution experiments and cleaves the intact pro-PO to an intermediate of 76kDa with no phenol oxidase activity, and then, PPAF-Ⅰ converts the intermediate to the active phenol oxidase of 60kDa in the presence of PPAF-Ⅱ. These results indicate that the activation of pro-PO system in hemolymph of H. dimophalia larvae is accomplished by least two activating factors, a serine protease and protein cofactor.

Molecular cloning of cDNA for pro-phenol-oxidase-activating factor Ⅰ, a serine protease is induced by lipopolysaccharide or 1,3β-glucan in coleopteran insect, Holotrichia diomphalia larvae

Lee, So Young,CHO, Mi Young,HYUN, Ji Hoon,LEE, Kwang Moon,HOMMA, Ko-Ichi,NATORI, Shunji,KAWABATA, Shun-Ichiro,IWANAGA, Sadaaki,LEE, Bok Luel 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

Previously, we identified two pro-phenol oxidase-activating factors, named PPAF-Ⅰ and PPAFⅡ, directly involved in the activation of the ourified pro-phenol oxidase(pro-PO) from hemolymph of the coleopteran,Holotrichia diomphalia larvae[Lee,S.Y.,Kwon,T.H., Hyun, J.H.,Choi,J. S. I., Iwanga,S,& Lee,B.L.(1998)Eur.J. Biochem. 254,90-97]. Here, we report molecular cloning of cDNA for PPAF-Ⅰ. Based on the sequence of the cloned cDNA, the PPAF-Ⅰ gene appears to encode a menber of serine protease zymogen consisting of 365 amino avid residue with a molecular mass of 40193 Da. The 109 amino acid residues preceding amino-trrminus Ile residue of the mature protein seem to constiture a prepro-sequence. The mature protein is a sreine protease composed of 256 amino acids with a calculated molecular mass of 28009Da. The overall structure is highly similar to that of Drosophila easter serine protease(42.9% identity),an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro-segment of PPAF-Ⅰwere similar to those of Tachhypleus proclotting enzyme and the mammalian neutrophil-derived defensin. Futjermore,[^3H]diisopropylphosphate(iPr_2P)-labled PPAF-Ⅰ was specifically produced from the crude preparation of PPAF-Ⅰ zymogen by incubation with lipopolysaccharide or 1,3-β-glucan, whereas [^3H]iPr_2P-labeled PPAF-Ⅰ was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro-PO-activation system in H. diomphalia larvae may proceed with the activation of PPAF-Ⅰ zymogen by microbial polysaccharides.

Activated phenoloxidase from Tenebrio molitor larvae enhances the synthesis of melanin by using a vitellogenin-like protein in the presence of dopamine

Lee, Kwang Moon,Lee, Kum Young,Choi, Hye Won,Cho, Mi Young,Kwon, Tae Hyuk,Kawabata, Shun-ichiro,Lee, Bok Luel 부산대학교 유전공학연구소 2000 분자생물학 연구보 Vol.16 No.-

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxdase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca^2+ and -1,3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 and β-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These data support the conlusion that the 160-kDa vitellogenin-like protein is involved in arthropod melanin synthesis.

Insecticidal activity of the metalloprotease AprA occurs through suppression of host cellular and humoral immunity

Lee, Seung Ah,Jang, Seong Han,Kim, Byung Hyun,Shibata, Toshio,Yoo, Jinwook,Jung, Yunjin,Kawabata, Shun-ichiro,Lee, Bok Luel PERGAMON 2018 DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY Vol. No.

<P><B>Abstract</B></P> <P>The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. <I>Pseudomonas entomophila</I> is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (<I>Riptortus pedestris</I>) that were injected with <I>P. entomophila</I> were highly susceptible to this bacterium. To determine how <I>P. entomophila</I> counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of <I>P. entomophila</I>. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus <I>Pseudomonas</I> by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of <I>P. entomophila</I>, we used an <I>aprA</I>-deficient <I>P. entomophila</I> mutant strain (Δ<I>aprA</I>). When Δ<I>aprA</I> mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the Δ<I>aprA</I> mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of <I>P. entomophila</I> via suppression of host cellular and humoral innate immune responses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>P. entomophila</I> showed high insecticidal activity against host <I>R. pedestris.</I> </LI> <LI> AprA, an alkaline zinc metalloprotease, is the major insecticidal protein of <I>P. entomophila</I>. </LI> <LI> AprA kills host <I>R. pedestris</I> by suppression of both host cellular and humoral innate immunity. </LI> </UL> </P>