i ABSTRAK PEPTIDA GLIADIN SEBAGAI PENINGKAT PERMEASI UNTUK PENGHANTARAN PROTEIN SUPEROKSIDA DISMUTASE Citrus limon PADA LAPISAN TUNGGAL SEL CACO-2 Oleh RATNA ANNISA UTAMI NIM : 307012012 ( Program Studi Doktor Farmasi ) Latar Belakang: Superoksida dismutase (SOD), merupakan suatu antioksidan kuat dan komponen penting dalam mekanisme pertahanan sel melawan tekanan oksidatif. Salah satu SOD potensial untuk aplikasi terapeutik adalah SOD dari lemon (Citrus limon). Namun, penghantaran SOD secara rute oral memiliki keterbatasan berupa ketersediaan hayati yang rendah akibat permeasi usus protein yang rendah dikarenakan ukuran protein yang besar. Salah satu pendekatan untuk mengatasi permasalahan permeasi usus yang rendah adalah dengan penggunaan senyawa peningkat permeasi. Tujuan: Penelitian ini bertujuan untuk mengkonstruksi fusi protein SOD Citrus limon (SOD_Cl) dengan peptida gliadin LGQQQPFPPQQPYPQPQPF (GliSOD_P51) dan QQPYPQPQPF (GliSOD_P61) untuk meningkatkan kapasitas permeasinya dalam menembus lapisan tunggal sel Caco-2, memperoleh ketiga protein rekombinan dengan kemurnian tinggi, mengkarakterisasi unit aktivitas spesifik, kestabilan terhadap pH dan suhu, parameter kinetik dan pengaruh penambahan senyawa kimia, menguji sifat sitotoksisitas dan kapasitas permeasinya menembus lapisan tunggal sel Caco-2, dan mempelajari mekanisme permeasi SOD_Cl, GliSOD_P51 dan GliSOD_P61 pada tingkat molekuler. Metode: Protein rekombinan SOD_Cl dan bentuk fusinya (GliSOD_P51 dan GliSOD_P61) dikonstruksi menggunakan pendekatan rekayasa genetik. Overproduksi ketiga protein rekombinan dilakukan pada Escherichia coli BL 21(DE3) dan dimurnikan menggunakan kolom afinitas Ni. Karakterisasi biokimia dilakukan dengan menentukan aktivitas residual SOD menggunakan metode oksidasi reduksi Nitro Blue Tetrazolium (NBT)-riboflavin. Uji sitotoksisitas dan permeabilitas dilakukan menggunakan kultur sel Caco-2. Sitotoksisitas ketiga protein rekombinan pada sel Caco-2 in vitro ditunjukkan dalam persentase viabilitas sel relatif terhadap kontrol negatif (tanpa perlakuan, 100%) yang ditentukan dengan uji Alamar Biru dan Tripan Biru. Pada uji permeabilitas, sel Caco-2 ditumbuhkan pada Transwell chambers hingga 6 dan 21 hari kemudian kapasitas permeasi ketiga protein rekombinan dan marka paraselular Fluorescein Isothiocyanate (FITC)-Dextran 10 kDa (FD 10) dan FD 40 ditentukan dengan menginkubasi protein dan FD secara bersamaan pada kompartemen apikal. Keberadaan ketiga protein rekombinan pada kompartemen apikal dan basolateral setelah uji permeabilitas dianalisis dengan elektroforesis

ii protein. Permeasi FD 10 dan FD 40 ditentukan dengan mengukur kadarnya pada larutan basolateral menggunakan spektrofluorometri. Studi mekanisme molekular peningkatan permeasi dilakukan dengan mengukur ekspresi gen ZO-1, Claudin-1, Claudin-2 dan Occludin menggunakan quantitative real-time PCR (qPCR). Hasil: Hasil penelitian menunjukkan bahwa tingkat ekspresi ketiga protein rekombinan tinggi yaitu sekitar 39,84%-42,38% dari total protein intraselular E. coli BL21(DE3). Tingkat kemurnian elektroforetik ketiga protein adalah lebih tinggi dari 95%. Hasil karakterisasi biokimia menunjukkan ketiga protein rekombinan memiliki kemiripan sifat biokimia termasuk stabil dan aktif pada rentang pH luas dari 2-8, relatif stabil (aktivitas residual lebih dari 80%) setelah inkubasi pada suhu di bawah 50°C dan relatif tahan terhadap senyawa kimia yang diuji pada konsentrasi rendah. Inhibisi oleh senyawa KCN dan H2O2 membuktikan ketiga protein rekombinan termasuk golongan Cu,Zn-SOD. Dari ketiga protein rekombinan, SOD_Cl memiliki aktivitas spesifik, nilai KM dan Vmax tertinggi, kestabilan suhu dan kestabilan pada pH basa paling tinggi. Hal ini menunjukkan bahwa penambahan peptida gliadin pada SOD_Cl menurunkan aktivitas spesifik, kestabilan terhadap suhu dan pH basa (9-11). Hasil pengujian sitotoksisitas menggunakan Alamar Biru dan Tripan Biru menunjukkan tidak adanya efek kerusakan yang disebabkan oleh ketiga protein rekombinan terhadap kultur sel Caco-2. Hal ini memastikan bahwa peningkatan permeabilitas bukan dikarenakan kematian sel. Berdasarkan hasil analisis SDS-PAGE, hanya protein GliSOD_P51 dan GliSOD_P61 yang terdeteksi dalam larutan basolateral menunjukkan bahwa kedua protein dapat menembus lapisan tunggal sel Caco_2 sementara SOD_Cl tidak dapat menembus. GliSOD_P51 dan GliSOD_P61 meningkatkan permeabilitas FD10 secara bermakna, namun tidak pada permeasi FD 40 dibandingkan dengan sel yang tidak diberi perlakuan atau kontrol negatif mengindikasikan peningkatan permeabilitas oleh kedua protein adalah melalui rute paraselular. Analisis ekspresi gen menunjukkan bahwa GliSOD_P51 meningkatkan permeabilitas paraselular tanpa melibatkan regulasi ekspresi gen claudin-1, claudin-2 ataupun occludin dan peningkatan ekspresi gen ZO-1, sedangkan GliSOD_P61 melalui penurunan ekspresi gen claudin-1, claudin-2 dan occludin tanpa melibatkan ekspresi gen ZO-1. Kesimpulan: Dalam penelitian ini, konstruksi plasmid rekombinan yang membawa DNA pengkode SOD_Cl, GliSOD_P51 dan GliSOD_P61 telah dilakukan. Tingkat ekspresi ketiga protein rekombinan pada E. coli BL21(DE3) dan tingkat kemurnian elektroforetik ketiga protein setelah pemurnian adalah tinggi. Karakterisasi biokimia menunjukkan ketiga protein rekombinan memiliki aktivitas tinggi, kestabilan terhadap pH, suhu, senyawa kimia dan kinetika enzimatik yang baik. Ketiga protein rekombinan bersifat tidak toksik terhadap sel kultur Caco-2. Studi permeabilitas in vitro menunjukkan bahwa peptida gliadin pada GliSOD_P51 dan GliSOD_P61 masing masing dapat meningkatkan kapasitas permeasi protein GliSOD_P51 dan GliSOD_P61 dan senyawa FD 10. Mekanisme peningkatan permeasi oleh GliSOD_P51 diduga terjadi pada tahap pasca translasi atau melibatkan protein TJ lain sedangkan GliSOD_P61 melalui penurunan ekspresi gen occludin. Secara keseluruhan hasil penelitian ini menunjukkan potensi penggunaan peptida gliadin pada GliSOD_P51 dan GliSOD_P61 sebagai sebagai peningkat permeasi yang bersifat tidak toksik. .

iii ABSTRACT GLIADIN PEPTIDE AS PERMEATION ENHANCER FOR PROTEIN DELIVERY OF SUPEROXIDE DISMUTASE Citrus limon IN MONOLAYER OF CACO-2 CELLS By RATNA ANNISA UTAMI Student ID : 307012012 (Doctoral Study in Pharmacy) Background: Superoxide dismutase (SOD), a potent antioxidant and is an important constituent of cellular defence mechanism against oxidative stress. One of potential SOD for therapeutic application is from Citrus limon. However, SOD delivery by oral route has limitation for its bioavailability caused by low intestinal permeation due to large size of protein. One approach to overcome this low intestinal permeation is by using permeation enhancers. Purpose : This research was intended to construct two fusion proteins of SOD Citrus limon (SOD_Cl) with gliadin peptides, LGQQQPFPPQQPYPQPQPF (GliSOD_51) and QQPYPQPQPF (GliSOD_61) to enhance their permeation through monolayer CaCO-2 cells, to obtain the three recombinant proteins with high purity, to characterize their specific unit activity determination, thermal and pH stability and stability in the presence of chemicals, their enzyme kinetics, also for their cytotoxicity and permeation capacity through monolayer CaCo-2 cells and to study the mechanism of SOD_Cl, GliSOD_P51 and GliSOD_P61 permeation at molecular level. Methods: The recombinant SOD_Cl and its fusions (GliSOD_P51 and GliSOD_P61) were constructed using genetic engineering approach. The three proteins were overexpressed in Escherichia coli BL21(DE3) and affinity purified using Nickel column. Biochemical characterizations were performed determining residual activity of SOD based on nitro blue tetrazolium-riboflavin oxidation-reduction. Cytotoxicity and permeability assay were conducted using Caco-2 cells. The cytotoxicity of the three SODs in Caco-2 cells in vitro was presented as a percentage of cell viability relative to the negative (without treatment, 100%) as determined using the Alamar Blue and Trypan Blue assay. For permeability testing, Caco-2 cells were grown in Transwell chambers until 6 and 21 days and permeation capacity of the three SODs and paracellular marker Fluorescein Isothiocyanate (FITC)-Dextran 10 kDa (FD 10) and FD 40 was evaluated by incubating proteins and FD together in apical compartment. The presence of proteins in apical and basolateral compartments after permeabiliy assay was analyzed by protein electrophoresis. The permeation of FD 10 and FD 40 was determined by measuring their concentrations in basolateral compartment using spectrofluorometry. Study of molecular mechanism of permeation enhancement was determined by measuring the expression of ZO-1, Claudin-1, Claudin-2 and Occludin genes using quantitative real-time PCR.

iv Results: Research result showed that the level of expression of the three proteins was high about 39.84% - 42.38% of total intracellular proteins of E. coli BL21(DE3). Their electrophoretic purity level was higher than 95%. Results of biochemical characterizations showed that the three proteins shared similar enzymatic properties including activity over a broad pH range from 2.0–8.0, were relatively stable (retained activity of more than 80%) after incubation at temperature below 50°C and relatively tolerant to chemical agents tested at low concentration. Inhibition by KCN and H2O2 confirmed that SOD_Cl belonged to Cu,Zn-SOD. Of the three proteins, SOD_Cl exhibited the highest specific activity and KM and Vmax values, and the highest thermal stability and stability at alkaline pH. This showed that the introduction of gliadin peptides to SOD_Cl decreased specific activity, thermal stability and stability at alkaline pH (9-11). Cytotoxicity assay results using Alamar Blue and Trypan Blue showed no deleterious effects caused by the three of SODs to the Caco-2 cells. This results suggest that the increase in permeability was not not due to cell deaths lead to high permeability. Based on SDS-PAGE analysis, only GliSOD_P51 and GliSOD_P61 proteins were present in basolateral solutions demonstrating that both proteins but not SOD_Cl was permeated across the Caco-2 cell monolayer. The GliSOD_P51 and GliSOD_P61 increased significantly permeability of FD10 but not FD 40 compared to cell treated with SOD_Cl or negative control indicating the increase of permeability by the two proteins was via paracellular route. Gene expression analysis showed that the GliSOD_P51 increased paracellular permeability without involvement the regulation of claudin-1, claudin-2 and occludin gene expression and the increase expression of ZO-1 gene, while the GliSOD_P61 by decreasing the gene expression of claudin-1, claudin-2 and occludin without involvement of ZO-1 gene expression. Conclusion: In this research, recombinant plasmid carrying DNA encoding for SOD_Cl, GliSOD_P51 and GliSOD_P61 are constructed and the three proteins are expressed in high level in E. coli BL21(DE3). The level of electrophoretic purity of the three recombinant proteins after purification is high. Biochemical characterization of the three recombinant proteins shows high activity, good thermal and pH stability and enzyme kinetics. All of three recombinant protein were not toxic to the Caco-2 cells. In vitro permeability studies showed that gliadin peptides in GliSOD_P51 and GliSOD_P61 increased the permeation capacity of proteins respectively and FD 10. The permeation enhancement mechanism by GliSOD_P51 appears to occur post-translationally or involving other TJ proteins while GliSOD_P61 through decreased expression of occludin gene. Taken all together, based on enhancement of permeation of paracellular marker and SOD proteins, and the results of molecular mechanism study shows that the potency of gliadin peptides in GliSOD_P51 and GliSOD_P61 as non toxic permeation enhancer.