Engineering actin-resistant human DNase I for treatment of cystic fibrosis.
Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.
Ulmer
J S
JS
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080, USA.
Herzka
A
A
Toy
K J
KJ
Baker
D L
DL
Dodge
A H
AH
Sinicropi
D
D
Shak
S
S
Lazarus
R A
RA
D000199:Actins / Q000494:pharmacology* / Q000648:ultrastructure; D003550:Cystic Fibrosis / Q000188:drug therapy* / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000648:ultrastructure; D004791:Enzyme Inhibitors; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013183:Sputum / Q000201:enzymology; D013329:Structure-Activity Relationship; D014783:Viscosity
D000199:Actins; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D004247:DNA; D003850:Deoxyribonuclease I