Revised Structure of the Active Form of Human Deoxyribonuclease IIα

MacLea, Kyle S.; Krieser, Ronald J.; Eastman, Alan

doi:10.1006/bbrc.2002.6687

Cited by 24 publications

(34 citation statements)

References 21 publications

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“…We concurrently examined the effect of the N-glycosylation inhibitor TM on both the size and activity of expressed DNase IIα. Cells that were transfected with pD2 exhibited a predominant band at 45 kDa, which was reduced in size to 37 kDa after treatment with TM ( Figure 1A), consistent with previous observations of DNase IIα as a glycoprotein [11,26]. As in our previous work [26], TM treatment greatly diminished, but did not completely ablate, acid endonuclease activity of this protein ( Figure 1B).…”

Section: Requirement Of the Signal-peptide Leader Sequence For N-glycsupporting

confidence: 91%

“…A mammalian expression vector generated in this laboratory containing the cloned cDNA of DNase IIα in the pcDNA 3.1(k) vector (Clontech, Palo Alto, CA, U.S.A.), designated pD2 [26], was used as the template for mutagenesis. Primers identical with the region surrounding each mutation site, incorporating the desired mutation, were designed and synthesized (Dartmouth Molecular Biology Core Facility).…”

Section: Experimental Vector Construction Of Dnase Iiα Site Mutantsmentioning

confidence: 99%

“…The pD2 vector containing the cloned cDNA of DNase IIα [26] was again used as the template for PCR amplification. Two truncation mutants were created : (i) ∆15, which deleted the nucleotides coding for the first 15 amino acids (the signal-peptide leader sequence) and replaced them with a new starting methionine residue, and (ii) ∆107, which deleted the region corresponding to the first 107 amino acids and replaced it with a methionine start site.…”

Section: Vector Construction Of Truncated Dnase Iiα Mutantsmentioning

confidence: 99%

“…Full-length porcine DNase IIα was reported to undergo a series of proteolytic cleavage events : the removal of a hydrophobic N-terminal signal peptide and further cleavage to produce a three-chain (α1\β\α2) structure [11,24]. However, recent work in this laboratory showed that active protein consists of one contiguous polypeptide, heavily glycosylated and containing intrachain disulphide cross-links [26]. Our results suggest that the previously proposed structure for the porcine protein is the result of degradation during protein purification from whole organs.…”

Section: Introductionmentioning

confidence: 99%

“…Our original attempts to produce the protein in bacteria were never successful at generating active protein (results not shown). In light of subsequent results suggesting the importance of glycosylation and disulphide bridging in activity [26], we investigated the use of a mammalian expression system for production, like that used in the production of recombinant human DNase I protein [28]. However, our previous results from transfection experiments in Chinesehamster ovary cells found that DNase IIα overexpression resulted in apoptosis over short time courses [21].…”

Section: Introductionmentioning

confidence: 99%

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

MacLea

Krieser

Eastman

2003

Biochemical Journal

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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.

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Section: Requirement Of the Signal-peptide Leader Sequence For N-glycsupporting

confidence: 91%

Section: Experimental Vector Construction Of Dnase Iiα Site Mutantsmentioning

confidence: 99%

Section: Vector Construction Of Truncated Dnase Iiα Mutantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

MacLea

Krieser

Eastman

2003

Biochemical Journal

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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

et al. 2008

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Plancitoxin I, the major lethal factor from the spines of crown-of-thorns starfish Acanthaster planci, is a 37 kDa protein composed of two different subunits, and it has potent hepatotoxicity. It is homologous with mammalian deoxyribonuclease II (DNase II) and exhibits DNase activity responsible for the hepatotoxicity. To obtain information on the structure-activity relationship of plancitoxin I, various mutants were expressed in Chinese hamster ovary cells and examined for DNase activity. The results with deletion mutants revealed the requirement of the signal peptide for the expression of intact plancitoxin I and the inability of each subunit to hydrolyze DNA. Mutation at the N-glycosylation site (Asn-274) did not reduce DNase activity, supporting the absence of carbohydrate moieties in the molecule. The mutant H303A exhibited no DNase activity, suggesting the importance of His-303 for the enzymatic activity. No DNase activity was detected in C29A, C169A, C318A and C337A, indicating that four Cys residues are critical to the enzymatic activity. However, DNase activity was completely maintained in C263A and somewhat reduced in C277A and C357A. Based on the results with the Cys-specific mutants, plancitoxin I was assumed to contain three disulfide bridges (29-169, 277-357 and 318-337) and one free Cys-263.Keywords Acanthaster planci Á Chinese hamster ovary cell Á Crown-of-thorns starfish Á Deoxyribonuclease II Á Expression Á Plancitoxin I

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Acid DNases and their interest among apoptotic endonucleases

Counis

Torriglia

2006

Biochimie

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Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated by its inhibitor, ICAD, which is cleaved by caspases. Other neutral DNases have been shown to cleave nuclear DNA in apoptotic conditions: endonuclease G, GADD. In cells, the cytosolic pH is maintained to 7.2, mostly due to the activity of the Na(+)/H(+) exchanger. In many apoptotic conditions, a decrease of the intracellular pH has been shown. This decrease may activate different acid DNases, mostly when pH decreases below 6.5. Three acidic DNases II are so far known: DNase II alpha, DNase II beta and L-DNase II, a DNase II, derived from the serpin LEI (Leukocyte Elastase Inhibitor). Their activation during cell death is discussed in this review.

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Revised Structure of the Active Form of Human Deoxyribonuclease IIα

Cited by 24 publications

References 21 publications

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

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

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

Acid DNases and their interest among apoptotic endonucleases

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