Cytosolic phospholipases A2 (cPLA2s) are enzymes that hydrolyze ester bonds at the sn-2 position of glycerophospholipids (simply termed phospholipids, hereafter) in a Ca2+ dependent manner and liberate 1-acyl-lysophospholipids and unsaturated fatty acids. Human cPLA2δ was first identified as a gene up-regulated in psoriatic skin. Murine cPLA2δ(mcPLA2δ was discovered in our research group by searching the mouse genome database by using relatively conserved regions within murine cPLA2α,β andγ as queries. cPLA2δ has a primary structure similar to cPLA2δ: a C2 domain on its N-terminus and a catalytic domain on its C-terminus. To explore the physiological and pathological roles of cPLA2δ, it is important to characterize biochemical properties including enzyme kinetics, substrate specificities, and effects of known PLA2 inhibitors using purified enzyme preparation.

Real time quantitative PCR analysis revealed that mcPLA2δ is exclusively expressed in placenta, testis and tongue epithelium layer. The anti-mcPLA2δrabbit polyclonal antibody was generated and the endogenous mcPLA2δ protein expression was examined. The endogenous mcPLA2δ protein was detected in placenta and tongue epithelium layer. The expression of mcPLA2δ was quite limited, which contrasted with cPLA2δ that is expressed ubiquitously. Together with a reported increase of human cPLA2δ expression in psoriatic skin, this tissue-specific expression pattern of mcPLA2δ suggests the inducible nature of cPLA2δ and its specific roles in respective tissues.

The recombinant mcPLA2δ was prepared from Sf9 cells utilizing (His)6-tag affinity chromatography and enzymatic properties of mcPLA2δ were characcterized. Since mcPLA2δ was originally identified as a cPLA2 paralogue, first, PLA2 activity of mcPLA2δ were examined. As expected, mcPLA2δexhibited PLA2 activity that requires mM of Ca2+ ion. As for substrate preferences, mcPLA2δ displayed PLA2 activity with a broad substrate specificity. To be more precise, mcPLA2δ did not show preferences for phosphatidylcholine over phosphatidylethanolamine and linoleoyl- over arachidonoyl- phospholipids in PLA2 activity.

Further analyses revealed the unexpected strong PLA1 activity of mcPLA2δ, which is about 100-fold higher than the PLA2 activity. By the action of PLA1 activity, phospholipids are hydrolyzed its ester bonds at the sn-1 position and saturated fatty acids and 2-acyl-lysophospholipids are released. mcPLA2δ showed a slight preference for phosphatidylethanolamine over phosphatidylcholine in PLA1 activity. The liquid chromatography-mass spectrometry (LC=MS) based PLA1 assay revealed that mcPLA2δhydrolyzes all the phospholipids examined, such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and phosphatidic acid, and produces 2-acyl-lysophospholipids. Several enzymes that exhibit PLA1 activity have been clones. However, cPLA2δdoes not show any sequence similarity with those PLA1 enzymes.

Recently, it is reported that 2-acyl-lysophospholipids are more potent than 1-acyl- lysophospholipids in the activation of some lysophospholipid G-protein-coupled receptors, including LPA6 and GPR55. Saturated fatty acids are reported to be involved in developing metabolic disorders and inflammation. Therefore, in contrast to previously-identified PLA2s, cPLA2δ produces 2-acyl-lysophospholipids and saturated fatty acids, and may play novel roles in vivo.

本研究はゲノムデータベースより同定された、マウス細胞質型ホスホリパーゼA2δ (mcPLA2δ) について、その生体内での機能を明らかにするため、その内在性の発現と、その性質について酵素学的解析を行ったものであり、下記の結果を得ている。

1.mcPLA2δの内在性の発現を検討した結果、RNAレベルの発現が確認されたマウスの胎盤と舌上皮において、mcPLA2δの内在性タンパク質の発現が確認できた。すなわち、mcPLA2δが生体内で機能し得ることが示唆された。

2.mcPLA2δタンパク質をSf9細胞にバキュロウィルスを用いて過剰発現し、組換えタンパク質としてmcPLA2δを精製し、精製酵素を用いて、mcPLA2δ の酵素活性を検討した。mcPLA2δはcPLA2のパラログとして同定された遺伝子であるので、PLA2活性とリゾホスホリパーゼ(LPL)活性について解析を行った。その結果、mcPLA2δはPLA2活性とLPL活性を持つ事が明らかになった。

3.mcPLA2δについて、PLA1活性を検討すると、PLA2活性やLPL活性より数十倍から百倍強いPLA1活性を持つ事が明らかになった。これにより、アミノ酸配列状はcPLA2に分類されるmcPLA2δは酵素学的にはPLA1と呼ぶべき酵素である事が示唆された。

4.mcPLA2δのPLA1活性についてLC-MS/MSを用いて基質特異性を検討した結果、ホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジン酸、カルジオリピンを基質とする事が示唆された。

5.ヒトと同様にマウスの乾癬モデルにおいてもmcPLA2δはその発現分布が誘導される事が明らかになった。

以上、本論文はmcPLA2δについて、マウス生体内に内在性のmcPLA2δがタンパク質として存在する事を初めて明らかにし、精製酵素を用いた酵素学的な解析からmcPLA2δがPLA1活性を主要な活性として持つことを明らかにした。本論文の成果はこれまで未知であったmcPLA2δの酵素活性を明らかにし、その生物学的意義を解明するための端緒となるデータを与えるものであり、乾癬など炎症疾患における脂質メディエーター産生とcPLA2δの役割についての解明に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。