Generation and characterisation of transgenic zebrafish expressing human alpha-1-antitrypsin
2017-02-22T02:56:35Z (GMT) by
The Z variant of α1-antitrypsin (Z-AAT) is the most common “severe” deficiency allele of α1-antitrypsin. It affects approximately 2% to 5% of Caucasians of European descent [1-3]. The Z mutation propels the intracellular linkages between Z-AAT monomers forming polymers which results in high levels of retention within the endoplasmic reticulum of hepatocytes . This hepatic accumulation has been associated with the development of Z-AAT-induced liver disease in some PiZZ patients. A collection of different tools is needed to help in the understanding of the exact role of α1-antitrypsin polymers in the development of Z-AAT-induced liver disease. In this thesis, mAbs specific to Z-AAT polymers were generated by using yeast derived Z-AAT as an antigen for injection into rats. The process of mAbs generation and screening led to the identification and characterisation of two unique mAbs 23A5 and 26D5. In particular, mAb 23A5 was found to preferentially bind to α1-antitrypsin polymers. The mAb 23A5 possibly binds a different epitope to mAb 2C1 that has been shown to recognise Z-AAT polymers formed in vivo. mAb 23A5 works in a range of applications such as indirect immunofluorescence, native polyacrylamide gel electrophoresis (PAGE) immunoblotting, denaturing PAGE immunoblotting, immunoprecipitation and immunohistochemistry. A zebrafish liver (ZFL) cell line expressing α1-antitrypsin was also developed in this thesis. Z-AAT in ZFL cells showed delayed secretion compared to wild type α1-antitrypsin (AAT). The expression of Z-AAT was associated with the development of cellular bodies in the cytoplasm of the ZFL cells. There was an increase in the number of ZFL cells displaying the cytoplasmic cellular bodies after heat treatment indicating that the cytomegalovirus promoter used to drive expression of α1-antitrypsin is responsive towards heat. There was evidence of Z-AAT polymers intracellularly and extracellularly in Z-AAT expressing ZFL cells. This indicates the formation of pathological polymers of α1-antitrypsin occurring in the ZFL cell system. Last, a transgenic α1-antitrypsin zebrafish model was developed to study the effects of hepatic accumulation of Z-AAT. The transgenic Z-AAT zebrafish develop “disease” phenotypes related to glycogen storage. The “glycogen” phenotypes were associated with high levels of hepatic glycogen and formation of vacuoles containing glycogen within the cytoplasm of the hepatocytes. Heat treatment of the Z-AAT transgenic zebrafish led to a change in the distribution pattern of hepatic Z-AAT resulting in a more perinuclear and cytoplasmic localisation, suggesting increased Z-AAT retention. There are two key findings from the α1-antitrypsin zebrafish model. First, there is evidence to suggest that transgenic α1-antitrypsin zebrafish maintain hepatic human AAT better than Z-AAT. Therefore, understanding the mechanism might be useful for therapeutic purposes for PiZZ patients. Second, variability was observed in Z-AAT transgenic zebrafish in that not all the fish developed the “disease” phenotypes. This observation is consistent with the PiZZ human condition where not all the patients develop Z-AAT-induced liver disease. Therefore, the transgenic α1-antitrypsin zebrafish model is a suitable model to study the effects of hepatic Z-AAT accumulation.