Structural requirements of human DNase II alpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging.
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.
MacLea
Kyle S
KS
Department of Pharmacology and Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755, USA.
Krieser
Ronald J
RJ
Eastman
Alan
A
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D017931:DNA Primers; D004220:Disulfides; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II