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A2405 - Effect of N-Glycosylation of Recombinant Alpha-1 Antitrypsin Therapeutic Protein Structure and Function: A Biophysical and Molecular Dynamic Simulation Study
Author Block: K. Karuppanan1, S. Alkanaimsh1, D. Silberstein1, A. Bernardi1, A. Guerrero2, Q. Wang2, C. B. Lebrilla2, R. Faller1, D. Kueltz3, J. Eiserich4, C. E. Cross4, S. Nandi5, K. A. McDonald1; 1Chemical Engineering, University of California, Davis, CA, United States, 2Department of Chemistry, University of California, Davis, CA, United States, 3Department of Animal Science, University of California, Davis, CA, United States, 4Department of Internal Medicine and Physiology of Membrane Biology, University of California, Davis, CA, United States, 5Department of Global HealthShare Initiative, University of California, Davis, CA, United States.
Alpha-1antitrypsin (AAT) is plasma protein which has a primary function of protecting lung tissues from neutrophil elastase induced inflammation. Its normal plasma concentration is 15-30 µM, making it the most abundant circulating antiprotease. A circulating serum level below 11 µM is an indication of alpha-1 antitrypsin deficiency (AATD), a genetic disorder affecting protein conformation,where point mutations causes misfolded or defective forms of the protein. The most severe point mutation is called the Z mutation (Glu342Lys), which causes the protein to misfold and accumulate in the endoplasmic reticulum of hepatocytes. AATD represents the most important genetic risk factor for chronic obstructive pulmonary disease (COPD), in which smoke-induced oxidation of a key methionine residue (Met 358) in AAT causes a loss of anti-elastase activity. In this study, We have produced a biobetter plant recombinant AAT (prAAT) using a transient expression system in Nicotiana benthamiana plants and developed an efficient downstream process for its purification using immunoaffinity resin. we characterized the site-specific N-glycosylation and molecular dynamics simulation of prAAT produced by transient expression in Nicotiana benthamiana plants. We obtained a 58±8% recovery of total prAAT using Alpha-1 Antitrypsin Select affinity chromatography resin, with the final purified prAAT showing over 98% purity and an isoelectric point of 4.6. We confirmed a single amino acid substitution of biobetter prAAT (methionine 358 to valine) in the reactive loop by mass spectrometry analysis and its function was confirmed by elastase inhibition assay. To obtain plant specific N-glycosylation of prAAT, secretion was targeted to apoplast by Ramy3D signal peptide. Site-specific N-glycosylation patterns analysis revealed that prAAT contain oligomannose-type, paucimannosidic-type and complex-type glycans. Using molecular dynamics simulation, we investigated the impact of N-glycosylation on prAAT structure and dynamics. Our observations reveal that the core structure of the protein remains intact regardless of N-glycosylation, but, there is significant variability in the conformation of the flexible N-terminal residues and the active loop. These conformational variabilities did not affect AAT function for elastase inhibition.