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Recombinant Alpha-1-Antitrypsin (AAT) ZZ Mutant Among Other Mutants Activity Against Different Serine Proteinase
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A7129 - Recombinant Alpha-1-Antitrypsin (AAT) ZZ Mutant Among Other Mutants Activity Against Different Serine Proteinase
Author Block: S. Kim; Konkuk University, Seoul, Korea, Republic of.
Recombinant α1-antitrypsin (AAT) ZZ mutant among other mutants activity against different serine proteinase
Siyoung Lee1,4, Areum Kwak1, Heijun Kim1,3, Sinae Kim1,2, Tam Thanh Nguyen1,2, Eunhye Kim1,2, Kwang Ha Yoo3, In Ae Kim3, Katsuyuki Takeda5, Edward D. Chan5, Xiyuan Bai5,
and Soohyun Kim1,3
1Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology; 2College of Veterinary Medicine; 3Division of Pulmonology, Department of Internal Medicine, School of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; 4YbdYbiotech research center, 402 Partners Tower 1, 83 Gasan digital 1-ro Geumcheon-gu Seoul 08589 Korea; 5Departments of Medicine and Academic Affairs, D509, Neustadt Building, National Jewish Health, 1400 Jackson St, Denver, CO 80206
Abstract
Introduction: Alpha (α)-1 antitrypsin (AAT) is a serine protease inhibitor and it is also known as serum trypsin blocker. AAT restrains a wide variety of proteases and protects inflammatory tissue destruction from enzymes, particularly neutrophil elastase (NE) and proteinase 3 (PR3), from inflammatory immune cells. AAT is mainly produced from liver and there have been numerous efforts to express an active form of recombinant AAT protein, but these trials have not been successful due to the glycosylation and secretion of recombinant AAT protein.
Methods: The full open reading frame of AAT in a mammalian expression vector failed to produce a soluble AAT protein though AAT has a hydrophobic signal sequence at N-terminus. Next, we fused Fc part of immunoglobulin to C-terminus of AAT in order increase solubility and stability of recombinant AAT protein. AAT-Fc protein was expressed in Chinese Hamster Ovary (CHO) cells because AAT has four potential glycosylation sites (NxS/T) crucial for its activity. Indeed, the large amount of recombinant AAT-Fc wild type (WT) and ZZ mutant including several other mutants AAT-Fc were sufficiently produced as soluble proteins.
Results: We examined recombinant AAT-Fc WT and the mutants’ abilities to impede the enzymatic activity of different serine proteinases. AAT-Fc ZZ mutant as well as other AAT-Fc mutants showed their ability to suppress serine proteinases and interestingly, an individual AAT-Fc mutant exhibited different degree of inhibitory activity against a particular serine proteinase.
Conclusion: The present study demonstrates for the first time the biological activity of AAT-Fc ZZ mutant among other AAT-Fc recombinant proteins. These functional and biochemical analysis of recombinant AAT-Fc inhibitory activity against serine proteinases may help to understand naturally occurring AAT mutations in different inflammatory disorders.
Keywords: α1-antitrypsin (AAT), Recombinant AAT-Fc, AAT-Fc ZZ mutant, Serine proteinase, Enzymatic activity
Author Block: S. Kim; Konkuk University, Seoul, Korea, Republic of.
Recombinant α1-antitrypsin (AAT) ZZ mutant among other mutants activity against different serine proteinase
Siyoung Lee1,4, Areum Kwak1, Heijun Kim1,3, Sinae Kim1,2, Tam Thanh Nguyen1,2, Eunhye Kim1,2, Kwang Ha Yoo3, In Ae Kim3, Katsuyuki Takeda5, Edward D. Chan5, Xiyuan Bai5,
and Soohyun Kim1,3
1Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology; 2College of Veterinary Medicine; 3Division of Pulmonology, Department of Internal Medicine, School of Medicine, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea; 4YbdYbiotech research center, 402 Partners Tower 1, 83 Gasan digital 1-ro Geumcheon-gu Seoul 08589 Korea; 5Departments of Medicine and Academic Affairs, D509, Neustadt Building, National Jewish Health, 1400 Jackson St, Denver, CO 80206
Abstract
Introduction: Alpha (α)-1 antitrypsin (AAT) is a serine protease inhibitor and it is also known as serum trypsin blocker. AAT restrains a wide variety of proteases and protects inflammatory tissue destruction from enzymes, particularly neutrophil elastase (NE) and proteinase 3 (PR3), from inflammatory immune cells. AAT is mainly produced from liver and there have been numerous efforts to express an active form of recombinant AAT protein, but these trials have not been successful due to the glycosylation and secretion of recombinant AAT protein.
Methods: The full open reading frame of AAT in a mammalian expression vector failed to produce a soluble AAT protein though AAT has a hydrophobic signal sequence at N-terminus. Next, we fused Fc part of immunoglobulin to C-terminus of AAT in order increase solubility and stability of recombinant AAT protein. AAT-Fc protein was expressed in Chinese Hamster Ovary (CHO) cells because AAT has four potential glycosylation sites (NxS/T) crucial for its activity. Indeed, the large amount of recombinant AAT-Fc wild type (WT) and ZZ mutant including several other mutants AAT-Fc were sufficiently produced as soluble proteins.
Results: We examined recombinant AAT-Fc WT and the mutants’ abilities to impede the enzymatic activity of different serine proteinases. AAT-Fc ZZ mutant as well as other AAT-Fc mutants showed their ability to suppress serine proteinases and interestingly, an individual AAT-Fc mutant exhibited different degree of inhibitory activity against a particular serine proteinase.
Conclusion: The present study demonstrates for the first time the biological activity of AAT-Fc ZZ mutant among other AAT-Fc recombinant proteins. These functional and biochemical analysis of recombinant AAT-Fc inhibitory activity against serine proteinases may help to understand naturally occurring AAT mutations in different inflammatory disorders.
Keywords: α1-antitrypsin (AAT), Recombinant AAT-Fc, AAT-Fc ZZ mutant, Serine proteinase, Enzymatic activity
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