The term calpain refers to a family of Ca2+ activated cysteine proteases. Among many different isoforms from various organisms, the mammalian μ- and m-calpains are considered as "typical" calpains and thus define the characteristics of what a calpain is. Despite many years of research, the activation mechanism and physiological functions of calpains are poorly understood. The Ca2+ requirement for activation of calpains is much higher than those observed in normal cells, and it is not well understood how these proteases can be activated in their natural habitat. Although many protein molecules have been identified as calpain substrates, the in vivo relevance of their interaction with calpain is unknown given that some of the fundamental properties of the enzyme remain unsolved. Knockout mice yielded limited information, either showing little to no defect or dying prematurely during the embryonic stages.

While most invertebrate calpains show significant deviations from the typical profile of calpains, those found in Drosophila melanogaster show significant homology and similarity to the typical mammalian calpains. Of the 4 calpain and calpain-related molecules identified, Drosophila Calpain A and Calpain B are highly resemblant of mammalian m-calpain, both in terms of structure and enzymatic behavior. Thus, they show promising signs that could make D. melanogaster an alternative organism for use in the calpain research.

While the striking similarities are undeniable, several differences also exist in what we currently know about the Drosophila calpain system. First, the two typical calpains of Drosophila lack a small subunit, which is known to be essential in mammals. Secondly, the endogenous calpain inhibitor, termed calpastatin in mammals, remains to be identified in Drosophila. Added to this, characterization of the enzymes themselves have not progressed enough to safely assume that the mammalian and Drosophila calpain systems are true parallels.

I have conducted studies to draw further parallels between the mammalian and Drosophila calpain systems. My efforts were concentrated on:

1)Characterization of Calpain B and interaction with Drosophila homologs of known calpain substrates,

2)Investigation of Calpain B in regard to formation of dimer/polymers and the mechanism of autolysis,

3)Identification of the Drosophila calpain inhibitor.

1)Characterization of Calpain B and interaction with Drosophila homologs of known calpain substrates

Calpain B was expressed recombinantly and enzymatic properties were investigated. As reported previously, Calpain B was activated by millimolar Ca2+ concentration. It was also autolyzed much like m-calpain. The active cysteine residue, predicted based solely on m-calpain sequence to be at position 314 in the full length enzyme, was mutated to serine and alanine and activity was completely abolished. Thus, structural and behavioral similarities between Calpain B and m-calpain is apparent from these results.

In order to find out whether Calpain B is similar to m-calpain in terms of substrate specificity, Drosophila homologs of well-known calpain substrates were recombinantly expressed. The degradation patterns of these proteins by both Calpain B and m-calpain were examined. The two patterns were nearly identical to each other for each of the tested substrates, and also agreed with the predicted degradation pattern from the known calpain cleavage sites in the mammalian substrates. Therefore, not only is Calpain B itself similar to m-calpain, but also shows similar substrate specificity, suggesting further parallels in their in vivo function. In addition, these results show that modification of the studied substrates by calpain cleavage has been conserved during evolution, adding much significance to the role of calpain in regulation of the substrates.

2)Investigation of Calpain B in regard to formation of dimer/polymers and the mechanism of autolysis

How Calpain B overcomes lack of a small subunit is examined in several ways. First, its natural molecular mass was examined using size exclusion chromatography and native polyacrylamide gel electrophoresis. Both methods suggested that the native molecular mass of active Calpain B is approximately 83 kDa, identical to that of monomeric Calpain B. Secondly, co-expression of 2 recombinant calpains having different affinity tags and subsequent purification methods targeting only one affinity tag at a time were used to see whether the 2 different recombinant calpains could be pulled down together. In case of dimeric interaction, purification by one affinity tag must pull down the other affinity-tagged calpain as well. However, this was not observed, and each purification method exclusively pulled down the intended affinity tags. The experiment was repeated using a mutant enzyme and an active enzyme having different tags, which allowed identification by activity. The result was identical. Therefore, Calpain B was found to be monomeric.

Mechanism of autolysis was studied using the inactive mutant and active Calpain B. In the mixture of the two, there was a significant delay in autolysis of the inactive mutant molecule, whereas the active enzyme was swiftly autolyzed. This suggests that the mechanism of autolysis is intramolecular, i.e.-self cleaving. Namely, only the active Calpain B underwent self cleavage (autolysis) in the early phase, and subsequently cleaved other molecules including the inactive mutant Calpain B.

3)Identification of the Drosophila calpain inhibitor

The Drosophila calpain inhibitor remains unidentified after 20 years of the first report describing its activity. There is no sequence in the Drosophila genome which resembles mammalian calpastatin, thus indicating the inhibitor may be significantly different in the amino acid sequence from the mammalian inhibitor. Purification of Drosophila calpain inhibitor was attempted using some of the known properties of the mammalian calpastatin. As reported previously the substance was heat-resistant, allowing removal of heat labile proteins by boiling. Further purification was done using Q-sepharose and size exclusion chromatography, results of which suggested that the molecule may be 50 to 80 kDa in molecular mass. Further purification was unsuccessful due to fragility of the inhibitor. From our results, however, the inhibitor appears to be a protein.

Taken together, the results shown in the present thesis show several new aspects of the Drosophila calpain system that parallel the mammalian counterpart. First, the properties of Calpain B resemble those of mammalian m-calpain. Second, Calpain B show similar substrate specificity to m-calpain. Third, Calpain B does not form homomeric complexes. Fourth, there are strong indications of existence of a proteinacious endogenous calpain inhibitor in Drosophila. Further, I have characterized autolysis of Calpain B to be an intramoleular event. With these promising signs, the D. melanogaster could be an ideal organism for conducting experiments for calpains, and may contribute to expanding our understanding of these enigmatic proteases.

本論文は5章からなる。第1章(chapter 1)はイントロダクションであり、カルパインに関する学問的背景と本研究の実験的背景を述べている。具体的には、哺乳類とショウジョウバエのカルパイン系に関する知見、特にこの二つの生物種のカルパイン系の共通点と相違点を述べ、さらに本研究を行う上で参考となるカルパインの構造と生化学的諸性質、及び内在性カルパイン阻害因子(カルパスタチン)などについて説明している。第2章(chapter 2)ではショウジョウバエのカルパインBの基本的な酵素学的性質、及び、基質特異性を検討している。まずカルパインBを大腸菌で発現・精製する実験系を構築している。次いでカルパインBの酵素学的性質を検討し、哺乳類の組織普遍的カルパインのひとつであるm-カルパインと類似していることを明らかにしている。中でも、活性化に必要なカルシウム濃度と自己消化のパターンの両方がm-カルパインと類似していることは重要である。さらに基質特異性を検討するため、既知の哺乳類カルパイン基質のショウジョウバエホモログに対するカルパインBの作用を解析している。基質の候補としてショウジョウバエのAtg5, gelsolin, FAK, p35を選び、GSTタグをN末端に付加して大腸菌で発現させた。これらの融合タンパク質を精製し、カルパインBを作用させたところ、ショウジョウバエカルパインBの切断反応によって、哺乳類カルパインが上記基質を切断した場合と同じような断片が生成した。これらの結果から、ショウジョウバエのカルパインBは哺乳類のm-カルパインと相同な基質特異性を持ち、類似した生理機能を果たしていることを推測している。第3章(chapter 3)ではショウジョウバエのカルパインBの二量体形成の可能性と自己消化機構の検討を行っている。哺乳類の組織普遍的カルパインには必須である小サブユニットがショウジョウバエゲノム中に存在しないため、ショウジョウバエのカルパインでは二量体形成によって小サブユニットを代替している可能性が以前は考えられていた。しかし、非変性PAGEやゲル濾過クロマトグラフィーでの挙動から、カルパインBは単量体として存在して活性を持つことを示している。さらに、異なるアフィニティータグを付加した2種のカルパインBを大腸菌の同じ細胞に共発現させ、一方のタグに対する精製をそれぞれ行った結果においても、ホモ二量体を作る相互作用はないことが示されている。また自己消化についても、活性のない変異カルパインBを、活性をもつカルパインBと共存させた反応系を用いて検討している。具体的には、活性をもったカルパインと不活性な変異カルパインを1:1の比率で存在させて自己消化反応を行ったとき、変異カルパインの自己消化だけが明らかに遅延することを見い出している。この実験の結果から、カルパインBの自己消化反応においては、分子内の切断反応が最初に起こり、その後、分子間の切断反応が起こることを示している。第4章ではカルパインの内在性阻害因子の存在を、ショウジョウバエにおいて検討した。哺乳類カルパスタチンのDNA情報を用いた相同性検索からは、ショウジョウバエのゲノム中に相同遺伝子を見出すことができないため、本研究では、カルパイン阻害活性を生化学的に同定することを試みている。ショウジョウバエ成体から得た粗抽出画分、その熱安定性画分、イオン交換(Qセファロース)及びゲル濾過クロマトグラフィーのそれぞれにおいて、カルパイン活性を阻害する物質を検出しているが、活性の不安定性などにより完全精製には至っていない。しかし、このカルパイン阻害因子の不安定性とゲル濾過における推定分子量(3~8万)から、ショウジョウバエにおいてもカルパインを阻害する内在性タンパク質が存在することを示唆している。第5章(chapter 5)では本論文全体に対する考察を行っている。具体的には、本研究の結論、カルパインの生理機能、これまでのカルパイン研究において不十分であった点などを述べ、本研究を含むショウジョウバエを用いたカルパイン研究の利点に関して考察している。特に本研究から得られた結果として、ショウジョウバエのカルパインBは構造上だけでなく、一般的な性質や基質特異性に関しても哺乳類組織普遍的カルパインに類似しているという結論が強調され、将来の研究方向が示されている。

なお、本論文第2章の一部と第3章は、榎森康文准教授との共同研究であるが、論文提出者が主体となって分析検証を行ったもので、論文提出者の寄与が十分であると判断する。

したがって、博士(理学)の学位を授与できると認める。