Ticks (Arthropoda: Ixodoidia) are notorious hematophagous ectoparasites and serve as a unique vector of various deadly diseases inflicting humans and animals. Among the ticks, the ixodid ticks or hard ticks are more common and widely distributed throughout the world. Hard ticks feed on blood-meals for a long time (5-10 days or more) making a large blood pool, the essential feeding lesion of ticks, beneath the host's skin. Prior studies suggest that hard ticks produce a vast array of pharmacologically active biomolecules that are injected into the feeding lesions during persistent blood-feeding processes, and play crucial modulatory roles in their feeding success. However, the precise molecular mechanism(s) that prevents blood coagulation and initiates fibrinolysis in the blood pool to facilitate successful acquisition of blood-meals is still unclear. This study was performed to characterize a novel cDNA encoding an EF-hand protein from the salivary glands of the ixodid tick, Haemaphysalis longicornis, and the protein has been named as longistatin. Biochemical and functional characterization revealed that longistatin plays crucial roles in the development and maintenance of blood pool by keeping the blood in a fluid state through out the entire feeding period of the ticks; thus, longistatin is essential for blood-feeding success of hard ticks and eventually for the survival of ticks.

Chapter 1: Identification of an EF-hand protein from the salivary glands of the ixodid tick, Haemaphysalis longicornis

Longistatin gene was cloned from the salivary-gland cDNA libraries of adult H. longicornis ticks. Sequence analysis revealed that the full-length longistatin cDNA consisted of 750 bases. The open reading frame (ORF) consisting of 471 nucleotides, extends from the residues 133-603 which codes for a protein of 156 amino acid residues having a calculated molecular mass of 17,788 Da and a pI of 4.84. The molecule has a signal peptide and is predicted to be cleaved at Ala21 -Gln22. The mature protein has a predicted molecular mass of 15,541 Da with a theoretical pI of 4.59. Longistatin contains two EF-hand Ca2+-binding domains at residues 83-94 and 135-146 and the domains conserve canonical structures. Longistatin shows distinct changes in its migration during electrophoresis through SDS-PAGE gels containing calcium or ethylenediaminetetraacetic acid (EDTA). Longistatin moves faster in the presence of Ca2+. Both recombinant and endogenous forms of longistatin can be stained with Rutheninum red, demonstrating that longistatin is a Ca2+-binding protein. Reverse-transcription PCR data showed that longistatin-specific transcript was expressed in all lifecycle stages of H. longicornis and was up-regulated only in blood-fed ticks. Organ-specific transcription analysis revealed a salivary gland-specific expression of the gene which peaked at 96-120 h of feeding but declined sharply as soon as they dropped off the host. Consistently, endogenous longistatin was localized in the salivary glands of ticks and also in feeding lesions at the site of attachment of ticks on the host, suggesting that longistatin is synthesized in, and is secreted from, the salivary glands and may have functional roles in the feeding processes of ixodid ticks.

Chapter 2: Enzyme kinetics of longistatin

In this chapter, the enzyme kinetics of the purified recombinant longistatin was determined. Although longistatin does not contain the conserved catalytic triad of typical serine proteases but it efficiently hydrolyzed several serine protease-specific fluorogenic, synthetic substrates. Among the serine protease-specific substrates, longistatin potently hydrolyzed those containing Arg at the P1 site, indicating its specific affinity for the amide bond of Arg. Catalytic rate was relatively high during hydrolysis of α-thrombin-specific substrate (KCat/Km 2.97 M-1s-1) followed by that of tissue-type plasminogen activator (t-PA)/urokinase-type plasminogen activator (u-PA) (KCat/Km 2 M-1s-1) and trypsin-specific substrate (KCat/Km 0.88 M-1s-1). Longistatin did not hydrolyze synthetic substrates specific for other groups of proteases. The enzyme was active at a wide range of temperature and pH, with the optimum at 37 °C and pH 7. Its activity was efficiently inhibited by various serine protease inhibitors such as, phenylmethylsulfonyl fluoride (PMSF), aprotinin, antipain and leupeptin with the estimated IC50 of 278.57 μM, 0.35 μM, IC50 41.56 μM and 198.86 μM, respectively. Additionally, longistatin was reacted with several cations; among them, Ca2+and Mg2+ slightly increased the enzymatic activity of longistatin and Zn2+ potently inhibited longistatin in a concentration-dependent manner with an IC50 value of 275 μM. Mn2+ also attenuated its functions but to a much smaller extent. The inhibitory effect of Zn2+ was completely revived by EDTA. Given that, these findings suggest that longistatin is a new potent atypical serine protease.

Chapter 3: Longistatin activates plasminogen and degrades fibrinogen of hosts

In the chapter 3, biological functions of longistatin were evaluated. To explore the biological functions, longistatin was incubated with several commercially available proteins related with the feeding of blood-meals from hosts, such as fibrinogen, plasminogen, thrombin, factor VIIa and factor Xa. The study revealed that longistatin (1.6 μM) potently degraded the α, β and γ chains of fibrinogen as it was done by plasmin (1.6 μM), and delayed fibrin clot formation. Longistatin was shown to bind with fibrin with the estimated Kd, Bmax and molar binding ratio (MBR) of 145.5±3.3 nmol/L, 3.1±0.6 μmol/L and 42.3±7.4, whereas those of t-PA were 159.2±7.4 nmol/L, 1.4±0.4 μmol/L and 19.3±4.7, respectively. Longistatin activated fibrin clot-bound plasminogen into its active form plasmin, as comparable to that of t-PA, and induced lysis of fibrin clot and platelet-rich thrombi. Longistatin induced more than 50% lysis of thrombi within 2 h at 640 nM concentration. Plasminogen activation potentiality of longistatin was increased up to 4 times by soluble fibrin. Taken together, these results suggest that longistatin may exert potent functions both as a plasminogen activator and as an anticoagulant in the complex scenario of blood pool formation.

Chapter 4: Longistatin is relatively resistant to plasminogen activator inhibitor-1

In this chapter, I have demonstrated that longistatin is resistant to Plasminogen activator inhibitor-1 (PAI-1) and activates plasminogen in the presence of PAI-1, and induces fibrinolysis. To determine the effect of platelet lysate on longistatin, I employed two-step indirect fluorogenic assays using plasmin-specific synthetic fluorogenic substrate (Boc-Glu-Lys-Lys-MCA). Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and two chain u-PA (tcu-PA) with the IC50 of 7.7 and 9.1 μg/ml, respectively, whereas for longistatin inhibition IC50 was 20.1 μg/ml (p<0.01). To explore the effect of PAI-1 on longistatin, I conducted direct fluorogenic assays using a t-PA-/u-PA-specific synthetic fluorogenic substrate (Pyr-Gly-Arg-MCA) since longistatin also hydrolyzed this substrate. Activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained its 76.73% initial activity even after 3 h of incubation with 20 nM of PAI-1. IC50 of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1, implying that longistatin is resistant to PAI-1 and may be an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.

Chapter 5: Effects of longistatin on blood feeding of ticks

To study the biological functions of longistatin directly in ticks, I conducted RNAi study by injecting longistatin-specific dsRNA into ticks through 4th coxa. All ticks microinjected with dsRNA were active and healthy during the incubation period. After placement on rabbit ears, all ticks of both treated and control groups actively attached. However, in the dslongistatin-injected group, 3 (2.5%) ticks were found dead at 72 h of attachment. All ticks in the dsmalE-injected group reached to repletion and detached by day 6 post-attachment. Notably, dslongistatin injection was shown to hamper the feeding of ticks. These ticks were poorly fed and most of them failed to engorge. Only two ticks (1.66%) dropped off the host following engorgement in the RNAi group. The mean body weight of the ticks collected after 6 days of feeding was significantly (P<0.01) lower in the RNAi-treated group (53.53±50.38 mg) than that of the control group (253.43±57.91 mg). A marked difference between blood pools induced by the ticks of RNAi-treated and control groups was observed. Large blood pools were developed at the site of attachment of each tick of the control group. Histologically, blood pools of control group were flooded with RBC but hemorrhagic changes were not detected at the biting areas of ticks of the RNAi-treated group, suggesting that the longistatin gene plays vital roles in the formation and maintenance of a blood pool as preceded by marked hemorrhage into tick-feeding lesions.

In conclusion, longistatin is synthesized in salivary glands, secreted with saliva and injected into host tissues during acquisition of blood-meals. Longistatin is essential for the development and maintenance of blood pool; thus, in the feeding processes and survival of hard ticks. Therefore, longistatin may be a potential target for the development of safe acaricide and effective anti-tick vaccine to control tick and tick-borne diseases.

吸血性節足動物のマダニは、獣医畜産領域において最も加害性の大きい外部寄生虫である。そのため、吸血の被害から動物を守るマダニ防除対策の成否は畜産物の生産性を大きく左右する。本論文では、マダニ吸血の分子機構に着目し、抗マダニ薬やマダニワクチンなどの新規マダニ防除対策を開発する上での標的分子と成り得るロンギスタチンの機能探索を実施し、マダニ吸血行動における役割を明らかにすることを目的として研究を行っている。

第1章では、国内最優占種マダニのフタトゲチマダニ成ダニ唾液腺cDNAライブラリーよりロンギスタチン遺伝子を分離し、塩基配列等の遺伝子解析の結果から、遺伝子1次、2次構造を明らかにしている。また、蛋白質として高次構造解析及び類似性検索から、ロンギスタチンは既存蛋白質データベースにはない新規の蛋白質であることを見出している。さらに、異種細胞でのロンギスタチン発現系を構築し、組換えロンギスチンの精製標品の作製に成功している。加えて、申請者が独自に構築したマダニ個体内での局在解析、発現変動を調べ、ロンギスタチンの産生挙動を明らかにし、カルシウムまたはエチレンジアミン四酢酸の存在下でロンギスタチンの蛋白質電気泳動での移動度に、2つのEF-hand Ca2+ bindingドメインが関与することを実証し、ロンギスタチンが分泌性のカルシウム結合蛋白質であることを明らかにしている。

第2章では、大腸菌発現の組換えロンギスタチンを用いて生化学的に酵素性状を解析している。各種加水分解解析系を構築して、定型セリンプロテアーゼ触媒部位が未保存であるロンギスタチンから、セリンプロテアーゼ特異的合成蛍光基質に対して顕著な加水分解活性を見出している。同時に、その活性は、セリンプロテアーゼ特異基質のP1部位に存在することを示し、至適温度およびpH、阻害剤試験などを重ねて、ロンギスタチンは非定型セリンプロテアーゼであると提唱するに至っている。

第3章では、繊維素溶解系におけるロンギスタチンの生理活性を検討し、プラスミンと同様に、フィブリノゲンのα、βおよびγ鎖の加水分解能を突き止め、同時にフィブリンとの結合能を確認している。また、ロンギスタチンには組織プラスミノゲンアクチベーター(t-PA)と同様に、フィブリン塊結合性のプラスミノゲンをプラスミンへと活性化させることにより、フィブリン塊や多血小板血栓を溶解することが見出されたことから、ロンギスタチンはt-PAと抗凝固因子の両方の機能を保有し、吸血部位の宿主皮下に構築されるblood pool(血液プール)の形成・維持に重要な役割を果たしていると結論づけている。

第4章ではさらに、ロンギスタチンの機能解析を実施している。プラスミノゲンアクティベーターインヒビター1(PAI-1)はt-PAと複合体を形成し、t-PAの除去によりプラスミンの産生が減少され繊維素溶解系が抑制されるが、ロンギスタチンはPAI-1に対する親和性が低く、PAI-1の存在下でもプラスミノゲンを活性化し、線維素溶解を惹起することを結合試験の結果から示している。実際に、20nM濃度PAI-1の存在下においても、ロンギスタチンはフィブリン塊を完全に分解させ、t-PAなど他のPAとは異なり、PAI-1の複合体が未形成であったことから、ロンギスタチンはPAI-1耐性のPAであると述べている。

第5章では、逆遺伝学的手法であるRNA干渉法を用いて、ロンギスタチンノックダウンマダニを作製し、これをウサギの耳に付着させてin vivo機能解析を行っている。ノックダウンマダニでは表現型として、著しい吸血阻害作用が惹起され、吸血開始6日後における対照群との平均体重の比較では、大きな有意差が認められ、結果的に吸血不全に陥ることが示されている。また、吸血部位の組織標本では、対照群で血液プール内に赤血球が充満しているのに対して、ノックダウンマダニでは出血性の変化を伴わない炎症性細胞の集積と血液プールの形成不全が惹起されることを見出している。

以上の結果より、唾液腺で作られるロンギスタチンは吸血時、唾液と共に分泌されて宿主組織皮下内に注入され、血液プール内において本研究で新たに発見された抗血液凝固経路を惹起させながら持続的かつ大量に宿主の血液を搾取可能になっていることを見出し、マダニ吸血行動において、ロンギスタチンは不可欠な分子であることを導き出した。以上の成果はマダニの生理・生態に立脚した安全で環境に優しい新たな抗マダニ薬の開発に結実するもので、化学的殺ダニ剤に全面依存した現行法に代わる新規防除技術の開発に大きく貢献するものである。よって、審査委員全員一致で本論文が博士(農学)の学位論文として十分価値があると認めた。