DNase IIα (EC 184.108.40.206) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3′-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure–activity relationship has been largely unexamined. Recently, we demonstrated that active DNase IIα consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415–421]. The present paper describes further work to examine the elements of DNase IIα protein required for activity. Truncated forms and site-specific mutants were expressed in DNase IIα-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase IIα contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase IIα that lacks the purported active-site His295 can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase IIα protein structure, which is important for three-dimensional structural analysis and for production of DNase IIα as a potential protein therapeutic for cystic fibrosis or other disorders.
Abbreviations used: CF, cystic fibrosis; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; NEB, New England Biolabs; PNGase F, peptide N-glycosidase F; SRED, single radial enzyme diffusion; TM, tunicamycin.
Present address: Cutaneous Biology Research Center, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, U.S.A.
The nucleotide sequence data for human DNase IIα and human DNase IIβ have been submitted to DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AF047016 and AF274571 respectively.