• BMB Reports
• /
• 제34권1호
• /
• pp.15-20
• /
• 2001

One of the innate immune reactions in invertebrates is the pro-phenoloxidase (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 biological 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 phenoloxidase (termed phenoloxidase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a C-terminal 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 bP 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. molitor larvae. This suggests that POIP might be engaged in the humoral defense reaction.

• Molecules and Cells
• /
• 제26권6호
• /
• pp.606-610
• /
• 2008

Phenoloxidase (PO), a melanin-forming enzyme around the foreign bodies, is an important component of the host defense system in invertebrates. Pro-PO is the enzymatically inactive zymogen form of PO. In the Drosophila genome, three Pro-PO isoforms have been identified to date. These include Pro-PO1 and 2, which are primarily expressed in crystal cells, and Pro-PO3, which is predominantly found in the lamellocytes. In this study, we demonstrated that Drosophila Pro-PO3, but not Pro-PO1 or 2, is enzymatically active in its zymogen form. These findings were evidenced by spectacular melanin forming capacities of various cells and tissues that overexpressed these pro-enzymes. Furthermore, the melanization phenotype observed in the lamellocyte-enriched $hop^{Tum-l}$ mutant was drastically reduced in the absence of PPO3, indicating that PPO3 plays a major role in the lamellocyte-mediated spontaneous melanization process. Taken together, these findings indicate that the biochemical properties, activation mode and in vivo role of Pro-PO3 are likely distinct from those of the other two Pro-PO enzymes involved in Drosophila physiology.

• BMB Reports
• /
• 제46권5호
• /
• pp.264-269
• /
• 2013

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-${\beta}$-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-${\beta}GRP$). Ap-${\beta}GRP$ was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-${\beta}GRP$ specifically bind 1,3-${\beta}$-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-${\beta}$-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-${\beta}GRP$ antibody could be recovered by addition of purified Ap-${\beta}GRP$. These results demonstrate that Ap-${\beta}GRP$ acts as a biosensor of 1,3-${\beta}$-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-${\beta}$-D-glucan or Ap-${\beta}GRP$ alone was unable to trigger the proPO system, but they both did. Ap-${\beta}GRP$ was specifically degraded following the activation of proPO with 1,3-${\beta}$-Dglucan. These results indicate the variation in the amount of Ap-${\beta}GRP$ after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response.

• 한국동물학회지
• /
• 제38권1호
• /
• pp.125-135
• /
• 1995

붉은지렁이 (Lumbricus rubellus)의 체내에 존재하는 prophenoloxidase-phenoloxidase(prPO$\longrightarrow$PO)의 활성계는 몇 종류의 다른 경로에 의해 활성화 됨을 발견하였다 Propo는 exogenous trypsin $\beta$ 1.3-glucan, Ca2' 이온. lipopolysaccharide (LPS) 및 열처리 등에 의하여 활성도가 증가 되었고 Ca2' 이온이 나머지 4가지 종류의 처리와 함께 병행되었을 때 그 효과가 더욱 증가하였다 Propo의 활성도는 LPS나 Ca2' 이온의 농도가 각각 1 5H 10-s g Lps/r리, 15 mM(Ca2')의 농도에서 propo의 최대활성치를 나타냈으나 그 이상의 농도에서는 propo의 활성이 오히려 감소하였다. LPS. $\beta$ 1,3-glucan 및 Ca2' 이온 등은 trypsin 억제인자인 soybean trypsin inhibitor(571)가 함께 존재할 경우 전혀 propo를 활성화 시킨지 못하는 것으로 미루어 $\beta$ 1,3-glucan 및 Ca2' 이온 등은 체내의 trypsin 유사 효소의 활성을 증가시켜 궁극적으로는 proPO$\longrightarrow$PO의 활성화 반응에 간접적으로 작용한다고 생각되었다. 한편. 571의 존재하에서도 50"C의 열처리는 propo의 활성화에 아주 효과적인 물리적 요인으로 작용하였다. 따라서 열처리는 Ca2'이나 LPS. f 1,3-glucan파는 달리 직접적으로 proPO$\longrightarrow$PO의 활성화 반응에 작용하는 것으로 생각되어 붉은 지렁이의 체내에서 proPO가 활성화되는 괴정(propo-activating system)에는 최소한 2가지 이상의 경로가 있다고 생각된다.생각된다.

• BMB Reports
• /
• 제41권2호
• /
• pp.102-107
• /
• 2008

Extracellular proteases play an important role in a wide range of host physiological events, such as food digestion, extracellular matrix degradation, coagulation and immunity. Among the large extracellular protease family, serine proteases that contain a "paper clip"-like domain and are therefore referred to as CLIP-domain serine protease (clip-SP), have been found to be involved in unique biological processes, such as immunity and development. Despite the increasing amount of biochemical information available regarding the structure and function of clip-SPs, their in vivo physiological significance is not well known due to a lack of genetic studies. Recently, Drosophila has been shown to be a powerful genetic model system for the dissection of biological functions of the clip-SPs at the organism level. Here, the current knowledge regarding Drosophila clip-SPs has been summarized and future research directions to evaluate the role that clip-SPs play in Drosophila immunity are discussed.

• 한국응용곤충학회지
• /
• 제42권4호
• /
• pp.353-360
• /
• 2003

곤충병원세균이 효과적으로 병원력을 발휘하는 데 대상 곤충의 면역저하가 요구될 수 있다. 본 연구는 대상곤충의 면역저하가 병원세균의 살충능력에 직접 연관된다는 가설을 세웠다. 곤충병원세균을 5령 누에(Bombyx mori)의 혈강에 접종하였을 때, X. nematophilus는 다른 곤충병원세균인 Staphyiococcus gallinarum 보다 1,200배 높은 병원력을 보였다. 비록, 두 곤충병원세균은 누에의 혈구세포에 대해서 독성효과를 가지고 있지만, X. nematophiius는 S. gallinarum과 비교해보다 빠르고 높은 세포독성 영향을 보였다. 세포성 면역반응에서 5${\times}$$10^{5}$농도로 세균이 접종되었을 때, 누에는 약 20개 정도의 소낭을 형성하였다. 특히, 살아있는 X. nematophilus가 접종된 누에에서 소낭 형성은 크게 감소하였지만, S. gullinarum은 소낭 형성 감소에 영향을 주지 않았다. 두 세균 모두는 prophenoloxidase (proPO)의 활성화를 억제시켰다. 그러나 X. nematophilus.가 S. gallinarum보다 높게 PO 활성화를 억제시켰다. 라이소자임의 활성유도는 S. gallinarum접종 후 4시간에서 관찰되었으나, X. nematophilus를 접종하였을 때는 라이소자임의 활성이 전혀 관찰되지 않았다 이러한 결과들은 누에에서 X. nematophilus가 S. gallinarum보다 더 큰 면역저하를 유발하여 병원성을 높였다는 것을 보여준다. 따라서 본 연구는 곤충병원세균에 의해서 유발된 면역저하는 곤충병원세균의 병원성과 연관되어 있다는 것을 제시한다.다.