Genetic, biochemical and preclinical studies on a tandem cluster of two human serpins: alpha-1-antitrypsin and serpina2

In my thesis I focused on novel aspects of alpha-1-antitrypsin (AAT or serpina1) and its close relative, serpina2 (alpha-1-antitrypsin related protein), a gene in physical proximity to the AAT gene. Put in a nutshell, in the first described project we found that serpina2 is not a pseudogene as previously suggested. In a recent publication on serpina2, a broad tissue distribution was reported and a protease inhibiting function for serpina2 was discussed, but specific analytical tools were not available at that time. The goal of this study was to develop appropriate tools and procedures to clarify the role of serpina2 as a potential modifier of inflammatory diseases. We used cDNA of many human tissues in qPCR experiments and found serpina2 mRNA exclusively expressed in the epididymis. In addition, we explored recombinant expression of serpina2 and established rat monoclonal antibodies, which were used for immune histology, Western blots and a sandwich ELISA. To search for potential target proteases we used fluorescence resonance energy transfer (FRET) substrates derived from the reactive center loop of serpina2 in activity assays. After transfection of HEK 293 cells, the vast majority of the protein remained inside in association with the host cells, while only small amounts of the normally secreted protein were found in the culture supernatant, suggesting that poor folding and aggregation propensity of serpina2 may contribute to cellular stress and tissue inflammation, like a well-known coding variant of serpina1. With our monoclonal antibodies we were able to detect serpina2 in epididymal tissue lysates of men. A serpina1 chimera, carrying the reactive center loop of serpina2, was cleaved by chymotrypsin suggesting chymotrypsin-like proteases are putative targets of serpina2. In conclusion, serpina2 is not a pseudogene; it is an epididymis specific potential inhibitor of chymotrypsin-like proteases and may improve sperm maturation, male fertility and reproductive success. In the second project, we characterized two common coding variants of AAT by comparing them in a hydrogen/deuterium exchange experiment combined with mass spectrometry. We revealed that a common single-amino acid variation is not functionally neutral, but affects the overall structural flexibility of the variants and their ability to fulfill their role as protease inhibitor. In the third project we describe a new method to improve storage of donor lungs by adding AAT to the perfusion and storage solution, which could allow extended storage and better preservation before implantation. Primary graft dysfunction and vascular damage of donor lungs immediately after blood reperfusion in the recipient increases with prolonged preservation times. Hence, cold ischemic storage for only six hours is generally accepted, after lungs are cooled down and conserved in an extracellular, colloid-based electrolyte solution. Natural AAT, a highly abundant human plasma proteinase inhibitor with additional putative functions in vivo, has been approved as a therapeutic in AAT deficiency patients. Using a realistic clinically oriented murine model of lung transplantation and adding AAT with a functional reactive center loop to a widely used preservation solution (Perfadex), we found that ischemic storage times of lung grafts at 4 degrees can be extended to 18 h with improved graft function after reperfusion in recipient mice. Double knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not those lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity. We conclude that the proteinase 3 and elastase inhibiting classical function of tissue AAT reduces the early reperfusion-related injury of transplanted lungs after extended ischemic storage, which makes it a promising strategy for improvement of organ preservation