Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

Abstract

DNase IIα (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 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.

Structural characterization of plancitoxin I, a deoxyribonuclease II-like lethal factor from the crown-of-thorns starfish Acanthaster planci, by expression in Chinese hamster ovary cells

“…This accounts for the lower generation of intact plancitoxin I in the co-transfection with Da and Db constructs than in that with the plancitoxin I precursor construct. It is interesting to note that human DNase II was first described as being composed of three different subunits [9], but this was subsequently revised to a single, contiguous polypeptide containing intrachain-but no interchain-disulfide bridges [12]. Despite this knowledge about human DNase II, the results with the co-transfection with Da and Db constructs support our previous finding that plancitoxin I is composed of a-and b-subunits linked by an interchain disulfide bridge [1].…”

“…However, the cells transfected with DSP showed no DNase activity, indicating that the signal peptide is indispensable for the expression of intact plancitoxin I. The requirement of the N-terminal signal peptide for the expression of intact enzymes also holds for mammalian DNase II members, as demonstrated by human and porcine DNase II [9,12]. As in the case of the DSP mutant, the four deletion mutants, Da, Db, D(SP ?…”

“…1), most of which are indeed glycosylated [10]. In addition, the carbohydrates attached to the N-glycosylation sites are known to be important for the catalytic activity of mammalian DNase II members [12]. On the other hand, plancitoxin I has only one Nglycosylation site at Asn-274 (Fig.…”

“…The structural features of human and porcine DNase II have been extensively examined, particularly in relation to enzymatic activity [9][10][11][12][13]. The results obtained so far can be summarized as follows: the signal peptide is required for the expression of active enzyme; individual subunits exhibit no enzymatic activity; N-glycosylations, specific His residues and disulfide bridges are indispensable to enzymatic activity.…”

Cloning and expression of deoxyribonuclease II from chicken

“…DNase II (EC 3.1.22.1) is an endonuclease with an acidic pH optimum, which has been reported in lysosomes and secretions from cells of many organisms (MacLea et al, 2003b;Evans and Aguilera, 2003). In humans and rodents, the mammalian organisms in which DNase II has been studied the most extensively, two members of the DNase II enzyme family have been identified: DNase IIα (Krieser and Eastman, 1998;Baker et al, 1998) and DNase IIβ (also known as DNase II-like Acid DNase or DLAD) (Shiokawa and Tanuma, 1999;Krieser et al, 2001).…”