Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Liao, Ta–Hsiu; Liao, Wen-Chieh; Chang, Hui-Chen; Lu, Kuo‐Shyan

doi:10.1016/0167-4781(89)90124-3

Cited by 37 publications

(31 citation statements)

References 25 publications

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“…5,[15][16][17] However, it is highly unlikely that lysosomal nucleases are responsible for the rapid turnover of plasmid DNA, since dissipation of the acidic pH of the endolysosomes with ammonium chloride or chloroquine 18,19 did not delay the disappearance of injected DNA (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer

et al. 1999

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Inefficient nuclear delivery of plasmid DNA is thought to activity of apoptotic and lysosomal nucleases; (2) disposal be one of the daunting hurdles to gene transfer, utilizing a of microinjected plasmid DNA was inhibited in cytosolnonviral delivery system such as polycation-DNA complex.depleted cells or following the encapsulation of DNA in Following its internalization by endocytosis, plasmid DNA phospholipid vesicles; (3) generation and subsequent elimhas to be released into the cytosol before its nuclear entry ination of free 3Ј-OH ends could be detected by the tercan occur. However, the stability of plasmid DNA in the minal deoxynucleotidyl transferase-mediated dUTP nick cytoplasm, that may play a determinant role in the transfecend-labeling assay (TUNEL), reflecting the fragmentation tion efficiency, is not known. The turnover of plasmid DNA, of the injected DNA; and finally (4) isolated cytosol, delivered by microinjection into the cytosol, was deterobtained by selective permeabilization of the plasma memmined by fluorescence in situ hybridization (FISH) and brane, exhibits divalent cation-dependent, thermolabile quantitative single-cell fluorescence video-image analysis. nuclease activity, determined by Southern blotting and 32 PBoth single-and double-stranded circular plasmid DNA disrelease from end-labeled DNA. Collectively, these findings appeared with an apparent half-life of 50-90 min from the suggest that the metabolic instability of plasmid DNA, cytoplasm of HeLa and COS cells, while the amount of cocaused by cytosolic nuclease, may constitute a previously injected dextran (MW 70 000) remained unaltered. We prounrecognized impediment for DNA translocation into the pose that cytosolic nuclease(s) are responsible for the nucleus and a possible target to enhance the efficiency of rapid degradation of plasmid DNA, since (1) elimination of gene delivery. plasmid DNA cannot be attributed to cell division or to the Keywords: gene transfer; plasmid DNA; turnover; degradation; DNase; microinjection Introduction Liposome-mediated cellular transfer of plasmid DNA is a promising approach for gene therapy. However, despite the significant amount of lipid/DNA complexes internalized by the target cells, transgene expression remains undesirably low.1 Obstacles to nuclear accumulation of plasmid DNA include: the slow internalization process of the lipid/DNA complex in certain cells; 2 the entrapment of DNA in the endolysosomal compartment; 1,3,4 and the diffusional barrier of the nuclear envelope. 5The underlying mechanism of escape of internalized plasmid DNA from the endo-lysosomes is not fully understood. This process involves the destabilization of the limiting membrane of the endolysosomal compartment, the dissociation of the lipid/DNA complex and the release of plasmid DNA into the cytosol.6-8 Penetration of naked plasmid DNA into the cytosol was verified by using the T7 polymerase transfection system, which Correspondence: GL Lukacs, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canad...

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Section: Resultsmentioning

confidence: 99%

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer

et al. 1999

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“…DNase II, known as a lysosomal enzyme (37)(38)(39), is observed in the cytoplasm of epithelial cells and also in the lens capsule. Strikingly, DNase II is highly concentrated in all fiber cell nuclei.…”

Section: Discussionmentioning

confidence: 99%

Involvement of DNase II in Nuclear Degeneration during Lens Cell Differentiation

Torriglia¹,

Chaudun²,

F³

et al. 1995

Journal of Biological Chemistry

107

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The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60, 23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.Apoptosis or programmed cell death occurs in many physiological and pathological situations where selection of cells is required (1-4). In 1980, a landmark study (5) revealed that glucocorticoids induced extensive DNA degradation in rat thymocytes in vitro at the onset of cell death. DNA cleavage occurred in a very specific pattern producing fragments of DNA that were multiples of 180 -200 base pairs. This indicated that the chromatin was cleaved at the linker DNA between nucleosomic cores. The characteristic ladder was first shown by Hewish and Burgoyne (6) To date, three different endonucleases have been involved in DNA fragmentation leading to nucleosomal appearance. Some authors, such as Peitsch et al. (8), claimed that the well characterized pancreatic deoxyribonuclease (DNase I) was constitutively expressed in cells of tissues potentially primed for apoptosis. This 30-kDa nuclease, active at neutral pH, could be responsible for DNA cleavage into oligonucleosomes during cell death. On the other hand, Barry and Eastman (9), studying apoptosis in Chinese hamster ovary cells, were unable to detect a Ca 2ϩ -Mg 2ϩ -dependent endonuclease. Instead, they identified another endonuclease, which was cation-independent, with optimal activity at pH 5. This enzyme was proposed to be DNase II (9, 10). Finally, Hughes and Cidlowski (11) showed a lower molecular weight nuclease, an 18-kDa peptide (termed NUC 18), which was activated by Ca 2ϩ and Mg 2ϩ and related to cyclophilin (12). This peptide appears as a novel enzyme whose activity correlates with apoptosis in thymocy...

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“…DNase II has been reported in lysosomes, nuclei and various secretions [4][5][6][7][8], and is critical to the process of mammalian development [9,10]. The activity of DNase II is distinguished from other endonucleases by its acidic pH optimum, the absence of a requirement for bivalent cations and its ability to hydrolyse double-stranded DNA to yield short oligonucleotides bearing 3h-phosphate groups, rather than 3h-hydroxyl groups.…”

Section: Introductionmentioning

confidence: 99%

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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Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Cited by 37 publications

References 25 publications

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer

Involvement of DNase II in Nuclear Degeneration during Lens Cell Differentiation

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

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