| Deoxyribonuclease IIα (DNase IIα) (EC 3.1.22.1) is an acidic endonuclease found in lysosomes and nuclei, and it is also secreted. Because little is known of the structural determinants of its endonuclease activity, the overall goal of this thesis was to investigate the protein structure and processing of human DNase IIα necessary to generate active enzyme. Two approaches were taken: examination of evolutionary conservation of critical residues and structure-function analysis of mutant protein forms.; Initially another human protein was cloned which had extensive homology to DNase IIα, termed DNase IIβ. The gene, found at 1p22.3, encoded a 357 amino acid protein with 39% identity to human DNase IIα but a restricted tissue expression pattern. Further database searches found 29 new homologs from 21 species, including the first non-metazoan homologs such as that from the bacterium Burkholderia pseudomallei. These homologs identified highly conserved residues that were predicted to be important in enzyme function.; DNase IIα purified from porcine spleen was previously shown to contain three peptides, two of which were bridged by a disulfide linkage, derived by processing of a single polypeptide. However, screening of human cell lines consistently demonstrated a 45 kDa protein size, even under reducing conditions. These results suggested that active DNase IIα consists of one contiguous polypeptide. Furthermore, truncated forms and site-specific mutations indicated that the signal peptide is required for correct glycosylation, and that N-glycosylation is important for formation of the active enzyme. However, enzymatic deglycosylation revealed that glycosylation is not required per se for DNase activity. In addition, mutations in any of six evolutionarily conserved cysteine residues completely ablated activity, consistent with disulfide bridging playing an important role in stabilizing the active protein conformation.; We created human DNase IIα expression constructs for secreted, inducible expression in Pichia pastoris and successfully generated active enzyme. This method of protein production allows both disulfide bridging and N-glycosylation. Initial activity comparisons between human DNase IIα and DNase I protein under conditions representative of cystic fibrosis mucus were suggestive of efficacy as a CF mucolytic agent. Future work is needed to improve protein yield and better evaluate its therapeutic utility. |
