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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.
Lee,Bok Luel,Lee,Kang Moon,Kim,Mi Hee,Choi,Hye Won,Cho,Mi Young,Kurata, Shoichiro,Natori, Shunji,Jang,Kyung Suk,Lee,Young Un The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.4
Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its NH₂-terminus was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.
Lee, Sun Woo,Lee, Bok Luel,Yoo, Mi Ae,Lee, Hyun Seong,Kim, Eun Jun 생화학분자생물학회 2002 BMB Reports Vol.34 No.1
One of the innate immune reactions in invertebrates is the praphenoloxidase (pro-PO) activation system that is involved in the generation of superoxide, melanin synthesis, and the subsequent sequestration of foreign matter entering the hemocoel of the invertebrates. However, the molecular mechanism of this biologics! reaction is still obscure. To expand our understanding of the biological roles of the pro-PO activation system in invertebrates, we performed a yeast two-hybrid screening by using three regions of pro-PO as bait and a yeast two-hybrid cDNA library from Tenebrio molitor larvae as prey We isolated a novel partial cDNA clone that encodes a glycine-rich protein that interacted with the active phenoloaddase (termed phenoloaddase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a Cterminal glycine-rich domain. The C-terminal glycine-rich domain showed sequential homology with those of insect antifungal proteins. Also, the yeast two-hybrid screen in a reverse orientation (using POIP as bait) yielded PO, suggesting that the PO-POIP interaction is specific. By using a 315 by PCR fragment of the N-terminal unique region of POiP, we cloned the full-length cDNA of POIP from the Tenebruo cDNA library constructed by using E. coli injected larvae. The interaction analysis between PO, and a truncated fragment lacking the N-terminal unique region of POIP, indicated that the N-terminal unique region is necessary for interaction between PO and POIP The expression level of the POIP mRNA is increased by bacterial injection into T. molilor larvae. This suggests that POIP might be engaged in the humoral defense reaction.
Lee,Bok Luel,Kim,Byeong Gee,Koo,Sun Hyang,Shin,Young Hee,Choi,Yun Lim,Choi,Su Kyung The Korea Science and Technology Center 2000 BMB Reports Vol.33 No.2
Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitors, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1,2 and 3(DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki=3.6×10-9M), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki=6.2×10-8M). Pre-treatment of the 33mg/㎏ of DI-mixture (active fractions from C18 open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.
Purification and Characterization of a Holotricin 1 Homologues from Holotrichia diomphalia Larvae
Lee, So Young,Lee, Young Un,Lee, Bok Luel 부산대학교 유전공학연구소 1996 분자생물학 연구보 Vol.12 No.-
An inducible antibacterial protein, named holotricin 1, from the hemolymph of immunized larvae of a coleopteran insect, Holotrichia diomphalia has been identified (Lee et al, 1995). This study reports that three antibacterial proteins with some amino acid residue differences, termed holotricin 1A, 1B and 1C, were purified to homogeneity from the hemolymph of immunized larvae of a coleopteran insect, H. diomphalia. The complete aminoacid sequences of holotricin 1A and 1B are one amino acid difference from that of holotricin 1. There is a difference of 13 amino acids in holotricin 1C compared to holotricin 1. These proteins consist of 43-amino acid residues with the same three disulfide linkage. The antibacterial activity of holotricin 1A and 1B against gram-positive bacteria shows that of similarity with holotricin 1. Holotricin 1C has low antibacterial activity compared to holotricin 1.
Lee, Jong-Ho,Kim, Na-Hyang,Winstel, Volker,Kurokawa, Kenji,Larsen, Jesper,An, Jang-Hyun,Khan, Adnan,Seong, Min-Young,Lee, Min Ja,Andersen, Paal Skytt,Peschel, Andreas,Lee, Bok Luel American Society for Microbiology 2015 Infection and immunity Vol.83 No.11
<P>The cell envelopes of many Gram-positive bacteria contain wall teichoic acids (WTAs). <I>Staphylococcus aureus</I> WTAs are composed of ribitol phosphate (RboP) or glycerol phosphate (GroP) backbones substituted with <SMALL>d</SMALL>-alanine and <I>N</I>-acetyl-<SMALL>d</SMALL>-glucosamine (GlcNAc) or <I>N</I>-acetyl-<SMALL>d</SMALL>-galactosamine (GalNAc). Two WTA glycosyltransferases, TarM and TarS, are responsible for modifying the RboP WTA with α-GlcNAc and β-GlcNAc, respectively. We recently reported that purified human serum anti-WTA IgG specifically recognizes β-GlcNAc of the staphylococcal RboP WTA and then facilitates complement C3 deposition and opsonophagocytosis of <I>S. aureus</I> laboratory strains. This prompted us to examine whether anti-WTA IgG can induce C3 deposition on a diverse set of clinical <I>S. aureus</I> isolates. To this end, we compared anti-WTA IgG-mediated C3 deposition and opsonophagocytosis abilities using 13 different staphylococcal strains. Of note, the majority of <I>S. aureus</I> strains tested was recognized by anti-WTA IgG, resulting in C3 deposition and opsonophagocytosis. A minority of strains was not recognized by anti-WTA IgG, which correlated with either extensive capsule production or an alteration in the WTA glycosylation pattern. Our results demonstrate that the presence of WTAs with TarS-mediated glycosylation with β-GlcNAc in clinically isolated <I>S. aureus</I> strains is an important factor for induction of anti-WTA IgG-mediated C3 deposition and opsonophagocytosis.</P>