Bioinformatic approach, cloning and enzymatic characterization of DNA transposons, piscine cathepsins and vertebrate PLC δ1-Lf

Abstract

본 연구는 어류를 포함한 척추동물에서 분자생물학과 생명정보학 기법을 이용한 새로운 유전자치료소재의 개발 및 질병관련 매커니즘 규명을 위해 새로운 비바이러스벡터를 위한 전이인자의 클로닝과 어류의 단백질 분해효소 및 기존에 밝혀지지 않은 새로운 척추동물의 신호전달관련 인지질가수분해효소를 동정하여 그 구조적 특징 및 효소의 특성을 분석하였다. 미꾸라지로부터 유래된 새로운 Tc1계열의 전이인자 (MMTS)들은 생명정보학적 기법을 통해 분석한 결과, 기존의 DNA 전이인자그룹과 다른 새로운 그룹을 형성하였으며, 새로운 비바이러스벡터로의 활용을 위해 MMTS 전이효소의 특성을 분석하였다. MMTS 전이인자는 미꾸라지 게놈에 약 0.027%를 차지하며, 역반복서열을 가진 단일 전이효소를 가지고 있다. 또한, BLAST 서열비교결과, Drosophila melanogaster, Xenopus laevis, Xenopus tropicalis와 Anopheles gambiae에서 높은 유사도를 나타내었다. 또한, 포유동물에서 질병관련인자 및 치료제로 많이 연구되고 있는 카텝신 및 억제유전자의 해양생물에서의 매커니즘규명 및 어병관련활용을 위해 생명정보학기법을 활용해 국내의 주요양식어종인 넙치를 대상으로 카텝신Z, F, S 및 카텝신 억제유전자 시스타틴 B와 시스타신 C유전자를 클로닝 및 재조합효소의 특성을 분석하고 조직별 발현양상을 비교하였다. 넙치의 카텝신 Z (PoCtZ)는 RT-PCR분석결과, 조직 전반적으로 분포하였으며, 지질다당질 (lipopolysaccharide; LPS)유도 후 조직별?시간별 발현분석결과 역시 비슷한 양상을 보였다. 재조합 넙치 카텝신 Z (proPoCtZ)는 glutathione S-transferase 복합단백질로 대장균 (E. coli) 57kDa의 분자량으로 발현되었으며, 최적 pH는 7.5, Antipain과 Leupeptin에 의해 활성억제되었다. 넙치의 카텝신 F 유전자는 LPS-유도 후 조직별?시간별 발현분석결과 근육에서 6 시간째에 과량발현양상을 보였다. 넙치 카텝신 F 재조합 단백질은 대장균에서 pGEX-4T-1발현벡터를 이용해 발현되어 합성기질인 Z-Phe-Arg-AMC로 활성측정하였다. 넙치의 카텝신 S 유전자 또한 클로닝되었으며, glutathione S-transferase 복합단백질로 pGEX-4T-1벡터를 이용하여 재조합단백질을 발현시켰다. 재조합단백질은 대장균 E. coli BL21 (DE3)균주에서 60kDa의 분자량으로 과량발현 및 정제되었다. 시스테인 단백질분해효소의 억제제인 시스타틴 유전자인 시스타틴 B (PoCystatin B)와 시스타틴 C (PoCystatin C)유전자는 각각 536 bp와 751 bp cDNA로 넙치에서 클로닝되었다. 재조합 넙치 시스타틴 B와 C는 E. coli BL21(DE3)pLysS균주에서 pEXP5-CT CT/TOPO？？ TA발현벡터스시템을 이용하여 11kDa과 14kDa의 재조합단백질을 생산하였다. 넙치의 카텝신과 시스타틴 유전자들의 위에 결과들에 비추어, 넙치의 카텝신과 시스타틴 유전자 및 단백질은 어류의 특이적인 특성들을 포함하지만 전반적으로 다른 포유류의 카텝신 및 시스타틴 유전자와 구조적, 효소특성 및 발현에서 많은 유사점을 가지고 있었다. 마지막으로, phospholipase C (PLC)는 10여가지 동위효소가 있으며, 이 PLC의 활성화는 세포의 성장, 증식, 대사, 분비 등에 관여하는 것으로 보고되어 왔으나, PLC δ동위효소들의 조절성 기작은 아직 불분명하다. 본 연구에서는 어류의 PLC δ1의 특성분석과정에서 휴먼을 포함한 척추동물에서 기존에 보고되지 않은 새로운 N-말단부위가 신장된 PLC δ1의 존재를 확인하였고, 마우스를 대상으로 유전체의 생물정보학적 분석을 통해 유추된 결과를 바탕으로 PLC δ1 Long form을 클로닝하고, 재조합효소를 생산하여 그 특성을 분석하였다. 그 결과, 포유류 및 어류의 PLC δ1유전자는 exon 3 (PH domain 포함부위)에서 exon 16 (3’UTR포함부위)까지는 동일하지만 새로운 Exon 2을 가진 N-말단부위를 가진 Long form과 기존의 짧은 말단부위를 가진 Exon 1에서 시작하여 Exon 3으로 전사 및 번역되는 Short form의 PLC δ1의 두가지 형태를 가지고 있음을 확인하였다. 마우스에서의 PLC-δ1Lf의 발현은 암컷 및 수컷 모두 조직 전반적으로 발현을 보였지만, 특히 위와 대장에서 과량발현을 보였다. 재조합 마우스 PLC-δ1Sf 와 PLC-δ1Lf 단백질은 유사한 PLC활성을 보였으나, 지질결합능에서는 PLC-δ1Lf단백질이 PS (phosphatidylserine)에서 강한 결합능이 나타났다.

Here, identification of novel proteins which are potential therapeutic targets was demonstrating that analysis of gene sequences using Genomics and Bioinformatic comparisons. Three different candidate group including a new subfamily of Tc1-related MMTS-like transposons for the application of new non-viral gene transfer vector system, piscine cathepsins and cystatins from olive flounder and novel vertebrate phospholipase C-δ1 Long form for the novel drug discovery targets in piscine and/or human diseases were bioinformatically identified A novel Tc1-like transposable element, MMTS, is identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. On the dot-hybridization analysis for measuring the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that MMTS transposon is present at about 3.36 × 104 copies per 2 × 109 bp, and accounts for approximately 0.027% of the mud loach genome. Novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fish have conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches using the MMTS consensus sequence showed significant elements from Drosophila melanogaster, Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae. These findings may indicate that MMTS-like transposons are uniquely evolved in fishes, and MMTS-like transposons comprise a new subfamily of Tc1-like transposons with D. melanogaster (foldback element FB4, HB2, HB1) and A. gambiae (Frisky). In this study, the cDNA encoding for cathepsin Z (PoCtZ) was cloned from the olive flounder, Paralichthys olivaceus. RT-PCR analysis revealed the ubiquitous expression of flounder cathepsin Z in normal and LPS-stimulated tissues. The cDNA encoding for the proenzyme of PoCtZ (proPoCtZ) was expressed in E. coli as a 57kDa fusion protein with glutathione S-transferase. Its activity was quantified via the cleavage of synthetic fluorogenic peptide substrates, and the optimum pH for the protease activity was 7.5. The recombinant proPoCtZ was inhibited by Antipain and Leupeptin. The cDNA of olive flounder (P. olivaceus) cathepsin F (PoCtF) was cloned by the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) approaches. The expression of the PoCtF gene was examined in various tissues of normal and LPS-stimulated flounder by RT-PCR compared with the inflammatory cytokines IL-1β, IL-6 and IL-8. The results of RT-PCR analysis revealed ubiquitous expression throughout the entirety of healthy flounder tissues; however IL-1β, IL-6 and PoCtF expressions increased significantly in muscle at 6h post-injection with LPS. The cDNA encoding mature enzyme of PoCtF was expressed in E. coli using the pGEX-4T-1 expression vector system. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC, a substrate commonly used for functional characterization of cysteine proteinases. The cDNA of P. olivaceus cathepsin S (PoCtS) was also cloned, and the cDNA encoding proenzyme of PoCtS was expressed in E. coli as a fusion protein with glutathione S-transferase in pGEX-4T-1 vector. The recombinant proPoCtS/pGEX4T1 was overexpressed in E. coli BL21 (DE3) as a 60kDa fusion protein. In this study, the 536 bp PoCystatin B and 751 bp PoCystatin C cDNAs were also cloned from olive flounder (P. olivaceus). The tissue-specific expression pattern of PoCystatin B and PoCystatin C were examined by RT-PCR. PoCystatin B and PoCystatin C were expressed in E. coli BL21(DE3)pLysS in pEXP5-CT CT/TOPO？？ TA Expression vector as 11kDa and 14kDa fusion proteins. According to these preliminary enzymatic analyses and mRNA expression analyses, together with the above mentioned structural characteristics, we can conclude that flounder cathepsins and cystatins are similar to that of the other mammalian cathepsins and cystatins including the piscine and novel gene-specific characteristics. During I proceeded to further understand the regulation and roles of N-terminal extended region of MlPLC-δ1, surprisingly novel exon 2 (Lf) located within the second intron region was found in all mammalian and fish transcripts from Bioinformatic approach. Sequence analysis of the genomic structure of mammalian and fish PLC-δ1 suggested that these transcripts share exon 3 (including PH domain) to exon 16 (3’UTR) at the same chromosomal position, but differ at the exon 1 (Sf) and novel exon 2 (Lf) of the transcripts. Based on mapping and sequence comparison, the well known PLC-δ1 (Short form, PLC-δ1Sf) and novel PLC-δ1 Long form (PLC-δ1Lf) gene structure was established. Here I also compare the expression analysis, identification and enzymatic characterization of two-types of mouse PLC-δ1 gene. Expression of mouse PLC-δ1Lf was ubiquitously distributed in tissues of male and female with high expression in the stomach and large intestine. As expected, mouse PLC-δ1Sf was also widespread in the tested tissues. The recombinant mPLC-δ1Lf and mPLC-δ1Sf proteins were expressed in E. coli, and the activity of recombinant mPLC-δ1Sf protein was shown higher than mPLC-δ1Lf protein. Although the general catalytic and regulatory properties of nove PLC-δ1Lf are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended phospholipase Cδ1 (PLC-δ1Lf) might reflect some distinctions in regulatory properties like tissue-specific expression, subcellular localization and/or lipid binding specificity especially in the PS (phosphatidylserine) between mouse PLC-δ1Lf and PLC-δ1Sf. Although the translated protein of PLC-δ1Lf transcript is still to be identified and characterized, the present study provides the basic foundation for future studies on the role of PLC-δ1s. Further studies are necessary on the involvement of an individual PLC-δ1Lf molecule in the function of N-terminal extended region, and promoter regulation assay of independent PLC-δ1Lf.