DNase II alpha (EC 3.1.22.1) 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 II alpha 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 II alpha
protein required for activity. Truncated forms and site-specific mutants were expressed in
DNase II alpha-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 II alpha 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 II alpha that lacks the purported active-site His(295) 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 II alpha
protein structure, which is important for three-dimensional structural analysis and for production of
DNase II alpha as a potential
protein therapeutic for
cystic fibrosis or other disorders.