Insect Immune Activation by Recombinant Apolipophorin-III and Its Application to Pest Control in Hyphantria cunea

Kang, Young-Jin,Kim, Hong-Ja,Seo, Sook-Jae 한국곤충학회 2003 Entomological Research Vol.33 No.4

Apolipophorin­III is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium. Recently, apolipophorin­III in Galleria mellonella larvae showed to play an unexpected role in insect immune activation. We identified the cDNA sequence of Hyphantria cunea apolipophorin­III by oligonucleotide­primed amplification, and 5'­and 3'­RACE PCR. Since H. cunea has an unusually low level of apolipophorin­III in the hemolymph, a recombinant apolipophorin­III was overexpressed using a baculovirus expression system to investigate its biological activity. Recombinant apolipophorin­III and E. coli were injected into the hemocoel of last instar larvae, and the changes in apolipophorin­III in hemolymph was determined by Western blot. Injection of apolipophorin­III induced a slight increase of apolipophorin­III in the recipients' hemolymph. While E. coli injection led to remarkably increased concentration of apolipophorin­III in hemolymph. To investigate the induction of antimicrobial peptide by the injection of recombinant apoLp­III and E. coli, Northern blot was performed. Apolipophorin­III injection as well as bacterial injection into the larvae showed the induction on the expression of lysozyme gene. Apolipophorin­III is apparently related to the immune response through an unknown mechanism. The role of apolipophorin­III in insect immunity should be related to the activation of transcription factor of antimicrobial peptide.

Cloning and blood-meal dependent induction pattern of apolipophorin-III from Anopheles sinensis

노미영,원란,조용훈,이용석,한연수 한국곤충학회 2009 Entomological Research Vol.39 No.6

Apolipophorin-III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood-meal on the expression pattern of apolipophorin-III in mosquitoes. This led us to clone and partially characterize the full-length cDNA of apoLp-III (AnsiApoLp-III) from Anopheles sinensis. Analysis of AnsiApoLp-III cDNA shows that the 728-bp sequence has a 582-bp protein-coding region with 94 bp of putative 5' untranslated region and 152 bp of 3' untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp-III has the highest identity (63%) to Culex quinquefasciatus apoLp-III. Temporal expression pattern analysis shows that although AnsiApoLp-III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp-III was induced in An. sinensis adult females after uptaking a blood-meal. Spatial expression patterns of AnsiApoLp-III shows that AnsiApoLp-III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp-III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood-meal influences AnsiApoLp-III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp-III in these organs.

Genomic organization, sequence characterization and expression analysis of Tenebrio molitor apolipophorin-III in response to an intracellular pathogen, Listeria monocytogenes

Ju Young Noh,Bharat Bhusan Patnaik,Hamisi Tindwa,Gi Won Seo,Dong Hyun Kim,Hongray Howrelia Patnaik,Yong Hun Jo,Yong Seok Lee,Bok Ruel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.

Knockdown of apolipophorin-III is caused disruption of mitochondrial membrane potential in Hyphantria cunea

Hong Ja Kim,Yong Il Kim,Yong Min Kwon,In Hee Lee,Byung Rae Jin,Yeon Soo Han,Hyang Mi Cheon,Young Jin Kang,Sook Jae Seo 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insects. Recently, apolipophorin-III in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of the apoLp-III in insects. To investigate the genes which have a relationship with apoLp-III in fall webworm larvae, we reduced endogenous Hc apoLp-III mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-III reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-III, which induced an elevated level of superoxide anion in Hyphantria cunea larvae. The observed effect of Hc apoLp-III RNAi suggests that Hc apoLp-III is related to the action/expression of antioxidants, especially MnSOD.

꿀벌부채명나방 성충 정소에 의한 아포리포포린-III의 흡수

윤화경(Hwa-Kyung Yun) 한국산학기술학회 2016 한국산학기술학회논문지 Vol.17 No.10

아포리포포린-III (apoLp-III)를 꿀벌부채명나방 종령 유충 혈림프로부터 KBr 농도구배 초원심분리와 겔크로마토그래피(Sephadex G-100)를 이용하여 분리, 정제하였다. KBr 농도구배 초원심분리가 끝난 후, 리포포린을 제외한 분획(lipophorin-free fractions)을 겔크로마토그래피 시료로 사용하였으며, 겔크로마토그래피를 행한후 sodium dodecyl sulfate(SDS)-전기영동으로 apoLp-III의 정제를 확인하였다. 또한, 본 연구에서는 유충 혈림프로부터 정제된 apoLp-III가 꿀벌부채명나방의 성충 정소에 의해 흡수되는 지를 조사하였다. 우화한 지 1일 또는 2일된 성충으로부터 성충 정소를 차가운 링거액에서 분리한 후 조직 배양의 시료로 사용하였다. 순수 정제한 apoLp-III를 dimethyl sulfoxide (DMSO)에 녹인 형광물질 fluorescein isothiocyanate (FITC)와 상온에서 계속 저어가면서 1시간 동안 배양하였다. FITC로 표지된 apoLp-III(FITC-labeled apoLp-III)를 Sephadex G-25 PD-10 column을 이용하여 정제하고 확인하였다. 정제된 FITC-labeled apoLp-III와 성충 정소 조직을 상온에서 30분간 배양하였다. 배양 후 형광현미경 (fluorescence Axioskop microscope)과 SDS-전기영동을 이용하여 FITC-labeled apoLp-III가 정소 조직으로 들어가는 지의 여부를 확인하였다. 그 결과 FITC-labeled apoLp-III가 성충정소로 흡수된다는 사실을 알 수 있었다. Apolipophorin-III (apoLp-III) was isolated and purified from the last larval hemolymph of Galleria mellonella by KBr gradient ultracentrifugation and gel chromatography (Sephadex G-100). After KBr gradient ultracentrifugation, the lipophorin-free fractions were used as the samples for gel chromatography. The purity of the finally purified apoLp-III was confirmed by SDS-PAGE after gel chromatography. In this study, we found that apoLp-III is taken up into the adult testes in Galleria mellonella. The testes were dissected from day-1 or -2 adults in cold Ringer"s solution and used for tissue culture. The protein moiety of apoLp-III was labeled with FITC dissolved in dimethyl sulfoxide (DMSO) at room temperature under conditions of continuous stirring for 1 h. The FITC-labeled apoLp-III was purified with a Sephadex G-25 PD-10 column. The tissues of the adult testes were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)-labeled apoLp-III. Fluorescein microscopy and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed that the adult testis tissues internalize the FITC-labeled apoLp-III. The results showed that apoLp-III is taken up by the adult testes.

꿀벌부채명나방 종령 유충 지방체에 의한 아포리포포린-III의 흡수

윤화경(Yun, Hwa-Kyung) 한국산학기술학회 2013 한국산학기술학회논문지 Vol.14 No.8

아포리포포린-III (apoLp-III)를 꿀벌부채명나방 종령 유충 혈림프에서 KBr 농도구배 초원심분리와 겔크로마 토그래피 (Sephadex G-100)를 이용하여 분리, 정제하였다. 본 연구에서는 아포리포포린-III가 꿀벌부채명나방의 종령 유충 지방체에 의해 흡수되는 지를 조사하였다. 종령 유충 지방체 조직을 FITC로 표지한 아포리포포린-III (FITC-apoLp-III)와 상온에서 30분간 배양하였다. 배양 후 형광현미경과 전기영동을 이용하여 FITC-apoLp-III가 지방체 조직으로 들어가는 지의 여부를 확인하였다. 그 결과 FITC-apoLp-III가 유충 지방체로 흡수된다는 사실을 알 수 있었다. Apolipophorin-III (apoLp-III) was isolated and purified from the last larval hemolymph of Galleria mellonella by the KBr gradient ultracentrifugation and gel chromatography (Sephadex G-100). In this paper, we examined that apoLp-III is taken up into the last larval fat bodies in Galleria mellonella. The last larval fat body tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)-labeled apoLp-III (FITC-apoLp-III). Fluorescein microscopy and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) revealed that the last larval fat body tissues internalize FITC-apoLp-III. The results show that the apoLp-III is taken up by the last larval fat body.

Identification and characterization of an Apolipophorin-III gene from Actias selene Hübner (Lepidoptera: Saturniidae)

Cen Qian, LeiWang,FangWang,Bao-Jian Zhu,Guo-Qing Wei,Sheng Li,Chao-Liang Liu,Lei Wang 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.1

Apolipophorin-III (ApoLp-III) is involved in lipid transport and innate immunity in insects. In this study, we isolated ApoLp-III gene (As-ApoLp-III) cDNA fromActias selene Hübner (Lepidoptera: Saturniidae). It contained 660 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. It showed similarities with ApoLp-III proteins from other insect species, especially Antheraea pernyi. The recombinant protein of As-ApoLp-III was expressed in Escherichia coli, and anti-As-ApoLp-III antibodies were prepared. The As-ApoLp-III gene was expressed at high levels in the fat body and integument, and at low levels in the hemolymph, testis, silk gland, malpighian tubule, ovary, and midgut. As-ApoLp-III gene can be upregulated by E. coli, Micrococcus luteus, Beauveria bassiana, and nuclear polyhedrosis virus (NPV) and showed a greater sensitivity for gram-negative bacteria and fungi than for gram-positive bacteria and virus. These data indicated that As-ApoLp-III may play an important role in innate immune responses of Actias selene.

Uptake and Localization of Apolipophorin-III in Ovary and Testis of the Common Cutworm, Spodoptera litura

Kim, Eung-Seok,Kang, Sung-Hoon,Choi, Chung-Sik,Kim, Beom-Su,Lee, Sang-Dae,Kim, Woo-Kap,Kim, Hak-R. 한국곤충학회 1999 Entomological Research Vol.29 No.3

Apolipophorin-III (apoLp-III) was isolated from the last instar larval hemolymph of Spodoptera litura by cation exchange chromatography and reverse phase chromatography. Molecular mass of apoLp-III was estimated to be 18,266 daltons. Presence and exact localization of apoLp-III in both ovary and testis were identified by immunological analysis. In the earliest stage of ovary, apoLp-III is confined to the electron dense granules in the sheath cells between oogonia. During vitellogenesis, apoLp-III in the hemolymph is actively taken up into ovary through intercellular spaces between follicle cells and then stored in protein bodies in the ooplasm. After apoLp-III is taken up from the hemolymph into the basal portion of the vas deference, it is localized to electron dense granules on the apical surface of the vas deference epithelium. In in vitro culture study, after apoLp-III is actively synthesized in adult fat body, most of the synthesized apoLp -III is secreted to the medium.

Purification and Properties of ApoLp-III from the Larval Hemolymph of Agrius convolvuli

Yun, Hwa-Kyung 한국곤충학회 2000 Entomological Research Vol.30 No.2

Apolipophorin-III(ApoLp-III)를 박각시나방(Agrius convolvuli)의 종령유충 혈림프로부터 gelpermeation chromatography (Sephadex G-100)와 ion exchange chromatography (DE-52)를 이용하여 분리, 정제하였다. 정제된 apoLp-III의 분자량은 12% SDS 전기 영동상에서 20 kDa로 측정되었으며, 또한, citcular dichroism spectrum측정에 의하여 상당한 양의 $\alpha$-helica1 2차 구조를 지니고 있는 것으로 밝혀졌다. Apolipophorin-III (apoLp-III) was isolated and purified from the last instar larval hemolymph of Agrius convolvuli by gel permeation chromatography (Sephadex G-100) and ion exchange chromatography (DE-52). The molecular mass of apoLp-III determined to be 20 kDa using 12% SDS polyacrylamide gel electrophoresis. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of $\alpha$-helical secondary structure.

Silencing of apolipophorin‐III causes abnormal adult morphological phenotype and susceptibility to Listeria monocytogenes infection in Tenebrio molitor

Bharat Bhusan Patnaik,Hongray Howrelia PATNAIK,박기범,조용훈,이용석,한연수 한국곤충학회 2015 Entomological Research Vol.45 No.2

Insect apolipophorin‐III is an exchangeable protein that is abundantly found in the hemolymph, and serves an important role in lipid transport, development, and innate immunity. In this study, we examined the role of apolipophorin‐III (TmapoLp‐III) during the adult eclosion stages of Tenebrio molitor by RNA interference (RNAi) analysis. After silencing of the mRNA transcripts at both larval and pupal stages, adult phenotypic defects were noticed. Defects included the incomplete shedding of pupal skin, shorter extension of the elytra, and improper folding of the hind wings. Most of the adults were malformed and died possibly due to dehydration. We also showed the involvement of TmapoLp‐III in conferring resistance to T. molitor larvae against Listeria monocytogenes infection. Mortality was found to be lower in non‐silenced intoxicated larvae while the TmapoLp‐III silenced larvae showed a significant susceptibility after 7 days post‐injection with a dose of 106 cfu/larvae.