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INTRODUCTION |
DNase II is an acid hydrolase located subcellularly in lysosomes
(1, 2) and has been shown to be reversibly associated with the
lysosomal membrane (3). The enzyme hydrolyzes DNA to 3'-phosphoryl
oligonucleotides in the absence of metal ions under acidic conditions
(4). Although DNase II activity can be detected in a variety of animal
tissues and body fluids (5), it has not been cited for association with
lysosomal storage diseases (6) or cancer (7). However, recent studies
have shown that DNase II may be involved in apoptosis in Chinese
hamster ovary cells (8), in lens cell differentiation (9), and in aging of rat brain (10).
Much of our understanding of the biogenesis of lysosomes comes from
biosynthetic studies of lysosomal enzymes in which phosphorylation of
mannose residues is responsible for the targeting (11). For the
biosynthesis of DNase II, which might help us understand more about
apoptosis, information is lacking. However, to investigate the
biosynthesis of DNase II, knowledge of its protein and cDNA structures is essential. To date only the overall subunit structure of
DNase II, which is a noncovalently linked
·
heterodimer, is
understood (12), and little is known about the gene organization of the
two subunits. Herein we report the primary structure determination of
DNase II and its cDNA nucleotide sequence. These should provide the
molecular and genetic bases for future studies on its involvement in
apoptosis and other physiological functions.
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EXPERIMENTAL PROCEDURES |
Materials--
DNase II was purified from porcine spleen
according to the method of Liao (12). Trypsin and chymotrypsin were
obtained from Worthington. Endoproteinase Lys-C and pepsin were from
Sigma. Plasmid pGEM-T and restriction enzymes were from Promega. The T7
sequencing Kit was from Amersham Pharmacia Biotech. The culture medium
for Chinese hamster ovary cells, the transfection reagent, primers for
PCR1 and DNA sequencing were
from Life Technologies, Inc.
DNase II Assays--
The hyperchromicity assay (13) was used
during purification of DNase II from porcine spleen. One unit of
activity is defined as the amount of DNase II necessary to cause an
increase of 1 absorbance unit, at 260 nm, per min in 1 ml of the assay
solution containing 0.04 mg of calf thymus DNA (Sigma), 0.01 M in EDTA and 0.15 M in sodium acetate, pH 4.6. The metachromatic agar diffusion assay method was according to Shen
et al. (14) with modification of the assay buffer.
Protein Sequencing--
Determination of amino acid
compositions, cleavages of polypeptides with proteases, and separation
of the resulting peptides on HPLC were essentially according to
procedures described previously (15, 16). The individual
and
subunits were prepared by gel filtration on Sephadex G-100 as described
previously (12). Peptides were sequenced on an Applied Biosystems
Sequencer (model 477A).
Disulfide Pairing--
Intact
subunit was digested with
pepsin in 0.1% trifluoroacetic acid, and the digest was analyzed on
HPLC. Cys-containing peptides were detected as cysteic acid-containing
peptides after performic acid oxidation. The pairing of Cys-containing
peptides was determined from amino acid compositions and peptide
sequences.
DNA Sequencing--
Total RNA was obtained from 0.5 g of
deep frozen porcine spleen by the guanidinium
thiocyanate-phenol-chloroform method (17), and a cDNA library was
synthesized from total RNA with the CapFinder library construction kit
(CLONTECH). The standard PCR was performed with 1 µg of cDNA and 10 pmol of primer. A DNA thermal cycler (Perkin-Elmer) was used to repeat the following cycle 35 times: 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. All PCR products were purified from agarose gel after electrophoresis. For
ligation with pGEM-T and cell transformation, the methods described by
Maniatis et al. (18) were used. For DNA sequencing, PCR
products were cloned into plasmid pGEM-T. The double-stranded DNA was
sequenced according to Sanger et al. (19) using the T7
sequencing kit (Amersham Pharmacia Biotech). All sequence homology searches were conducted using the MPsrch program (Intelli
Genetics).
Construction of Expression Plasmid--
The cDNA fragment,
amplified by PCR with two specific primers
(5'-CGGGATCCTAGACCTTTAGCTGTATG-3' and 5'-ACTGAAGTCTGAATTCGCCCCTGAG-3'), covered the entire reading frame from the initiation to the stop codon.
The PCR product was, after treatment with BamHI and
EcoRI, ligated into pcDNA3 plasmid (Invitrogen). This
inserted plasmid was transformed into Escherichia coli
strain DH5
for initial cloning and for subsequent maintaining of the
cloned expression plasmid, pcDNaseII. The fidelity of the expression
plasmid was verified by restriction mapping and DNA sequencing.
Gene Expression--
10 µg of pcDNaseII in 100 µl of
serum-free medium was mixed with 20 µl of LipofectAMINE reagent (Life
Technologies, Inc.). 30 min after mixing, the mixture was used to
transfect Chinese hamster ovary cells, which were prewashed twice with
serum-free medium. 12 h after transfection, cells were washed
twice with serum-containing medium and incubated in the same medium.
After incubation, cells were harvested and separated from the medium by
centrifugation and lysed in 200 µl of 50 mM sulfuric acid
by freezing and thawing. All incubations were at 37 °C in a
CO2 incubator. The cell extract (lysate) and growth medium
were assayed for DNase II activity.
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RESULTS |
The Three-chain Structure--
When treated with 8 M
urea, DNase II dissociates into
and
subunits that can be
separated by gel filtration on Sephadex G-100 (12). These two subunits
also separate on reversed phase HPLC (Fig.
1a). However, after disulfide
cleavage by reduction and S-carboxymethylation, the
subunit is resolved into two components (Fig. 1b),
indicating that it consists of two chains connected by disulfide(s).
This two-chain structure for the
subunit is supported further by
the finding that
subunit yields two principle phenylthiohydantoin-derivatives, PTH-Leu and PTH-Ser, in the first cycle of protein sequencing. These two polypeptides are thus designated as the
1 and
2 chain, respectively.

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Fig. 1.
Three-chain structure of DNase II.
Panel a, separation of and subunits. 20 µg of
purified DNase II in solvent A (0.1% trifluoroacetic acid) was
injected. Panel b, separation of RCm- 1 and RCm- 2
chains. The subunit peak fraction in panel a was
lyophilized and suspended in 40 µl of 8 M urea containing
0.15 M Tris-HCl, pH 8.8, with 40 mM
-mercaptoethanol. After 10 min, the reduced polypeptide was
alkylated with 2 µl of 1 M iodoacetate, and the solution
with the addition of 500 µl of solvent A was injected
immediately. HPLC conditions: column, Vydac 218TP54 C18, 300 Å (4.6 × 250 mm), preequilibrated in solvent A; elution, a linear
gradient of 0-100% solvent B (80% acetonitrile containing 0.08%
trifluoroacetic acid) in 25 min; flow rate, 1 ml/min. The
identity of each peak was confirmed by protein sequencing.
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Amino Acid Sequence--
The amino acid sequences of
1,
,
and
2 chains of DNase II, determined by protein sequencing, are
shown in Fig. 2. These sequences bear no
homology to any of the sequences of DNase I (20) or any other protein
of known function, based on a sequence homology search in the GenBank
data base. Isolation and identification of all peptides to substantiate
the elucidated sequence are shown in Figs.
3-6.

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Fig. 2.
Complete amino acid sequences of the three
polypeptide chains of DNase II. Designation of peptides:
T, trypsin; C, chymotrypsin; Th,
thermolysin; L, endoproteinase Lys-C. The previously
determined (12) His active site peptide, ATEDHSKW, is
2(184-191).
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Fig. 3.
HPLC profile for the thermolytic digest of
RCm- 1 chain. Approximately 5 nmol of RCm- 1 chain (see Fig.
1b for its preparation) in 0.1 ml of 0.1 M
Tris-HCl, pH 8.0, was digested for 12 h with thermolysin. The
digest with the addition of 0.4 ml of 0.1% trifluoroacetic acid was
injected. HPLC conditions: column, Nova-Pak (3.9 × 150 mm,
Waters); elution, a linear gradient of 0-80% acetonitrile with 0.1%
trifluoroacetic acid; flow rate, 1 ml/min; temperature, ambient.
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Fig. 4.
HPLC profiles for the tryptic and
chymotryptic digests of subunit. Approximately 5 nmol of subunit in 0.1 ml of 0.1 M Tris-HCl, pH 8.0, was digested
for 12 h with trypsin (panel a) or chymotrypsin
(panel b). The digests with the addition of 0.4 ml of 0.1%
trifluoroacetic acid were injected. HPLC conditions are the same as
those described in the legend of Fig. 3.
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Fig. 5.
HPLC profiles for the endoproteinase Lys-C
and tryptic digests of RCm- subunit. Approximately 5 nmol of
RCm- subunit in 0.1 ml of 0.1 M Tris-HCl was digested
for 12 h with Lys-C at pH 8.8 (panel a) or trypsin at
pH 8.0 (panel b). The digests with the addition of 0.4 ml of
0.1% trifluoroacetic acid were injected. HPLC conditions are the same
as those described in the legend of Fig. 3.
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Fig. 6.
HPLC profiles for the chymotryptic digest of
RCm- subunit. Approximately 5 nmol of RCm- subunit in 0.1 ml
of 0.1 M Tris-HCl, pH 8.0, was digested for 12 h with
chymotrypsin. Panel a, gel filtration profile of the digest
on Sephadex G-25 (column size, 8 × 300 mm; fraction size, 1 ml;
eluent, 0.1% trifluoroacetic acid; flow rate, 3 ml/h). HPLC profiles
in panels b, c, d, and e
are for fractions 6, 7, 8, and 9 of the gel filtration, respectively.
HPLC conditions are the same as those described in the legend of Fig.
3.
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Disulfide Pairing--
Because seven half-Cys residues are present
in the
2 chain and one in
1 (Fig. 2), only one interchain
disulfide between the two chains is possible. The other six half-Cys
must form three intrachain disulfides in
2. The interchain disulfide
is
1Cys3-
2Cys52, confirming the finding
that the
subunit consists of
1 and
2 chains connected by
disulfide(s) (Fig. 1b). The three intrachain disulfides are
2Cys124-
2Cys192,
2Cys160-
2Cys240, and
2Cys201-
2Cys220. Peptides for disulfide
pairing are identified as shown in Fig. 7.

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Fig. 7.
HPLC profile for the peptic digest of intact
subunit. Approximately 6 nmol of intact subunit in 0.1 ml
of 0.1% trifluoroacetic acid was digested with pepsin for 12 h.
The digest was then injected. HPLC conditions are the same as those
described in the legend of Fig. 3 except that the column used was a
Vydac 218TP54 C18, 300 Å (4.6 × 250 mm).
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Sugar Attachment Sites--
Six potential
N-glycosylation sites are present in DNase II, two in
and four in
. Sugar analyses of the peptides containing these sites
show that each peptide contains 1.5-1.8 residues of glucosamine and
3.5-5.3 residues of mannose (Table
I).
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Table I
Sugar compositions of the N-linked glycopeptides
Glycopeptides were hydrolyzed in 100 µl of 2 M
trifluoroacetic acid for 4 h at 100 °C. Sugars were analyzed on
a Dionex sugar analyzer (model DX500) equipped with a pulsed
amperometric detection system (21).
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The cDNA Sequence--
A unique 0.2-kilobase DNA fragment was
obtained by PCR using the cDNA library as template with a
5'-degenerate primer, 5'-TAYCCNATGGTNTAYGAYTA-3' (Y = C or T;
sense; DNase II
71-76), and a 3' degenerate primer, 5'-AGRTCRTCNCCRAARTTNCC-3' (R = A or G; antisense; DNase
II
145-150). Direct sequencing of this PCR product shows identity of
the translated amino acid sequence with that determined by protein
sequencing. Based on this nucleotide sequence, a 5'-specific primer,
5'-CCTCCAGGAACCCTGGAACAGC-3' (sense; DNase II
100-106), and a
3'-specific primer, 5'-GGGCCAGGAAAGGCTATCTGGG-3' (antisense; DNase
II
170-177), were synthesized for 5'- and 3'-rapid amplification of
cDNA end reactions. The 5'- and 3'-end fragments obtained were
sequenced. Together they covered the entire coding region with a large
overlap. Partial restriction map and sequencing strategy are
illustrated in Fig. 8. The deduced
cDNA sequence is a 1,292-base pair polynucleotide that includes a
5'-untranslated region, the coding region, and a 3'-untranslated region
followed by a poly(A) tail (Fig. 9).

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Fig. 8.
Partial restriction map and sequencing
strategy for a DNase II cDNA clone. Arrows indicate the
extent and direction of the sequencing reactions. All regions are
covered by sequencing from both directions. bp, base
pairs.
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Fig. 9.
Nucleotide and its encoded amino acid
sequence for a cDNA of DNase II. The initiation and stop
codons are in boldface. The overlap region of the 5'- and
3'-end fragments is marked by asterisks. The open reading
frame-encoded amino acid sequence is shown below the nucleotide
sequence. The truncated peptides are in lowercase letters;
N-glycosylation sites are shaded; half-Cys
residues are boxed. This cDNA sequence has been deposited in
the GenBankTM/EDI data bank and is available under the accession number
AJOD1387.
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Expression of DNase II Activity--
Based on the sizes of the
diffused dark rings (Fig.
10a), DNase II activity was
estimated to be 0.1 units/ml in the growth medium with very little in
the cell extract, 72 h after transfection. The presence of DNase
II activity in the growth medium of transfected cells was also
confirmed using the plasmid DNA degradation assay; 15 min after
incubation the substrate (plasmid DNA) was completely degraded (Fig.
10b). DNase II activity was also detected, in lesser amounts, in the growth medium 48 h after transfection.

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Fig. 10.
Assays for DNase II activity in the cell
extract and growth medium fractions of Chinese hamster ovary cells
72 h after transfection. Panel a, metachromatic agar
diffusion assays. Each well contained 10 µl of sample.
Photographs were taken approximately 8 h after the application of
sample. Panel b, plasmid DNA degradation assays. The
reaction mixture (15 µl) contained 10 µl of growth medium and 0.5 µg of plasmid DNA (pcDNA3) in 0.3 M sodium acetate,
pH 4.7, and 10 mM EDTA. Incubation was at 25 °C. 5 µl
of 0.5 M Tris-HCl, pH 8.0, was added at the indicated times
to terminate the reaction, and the entire content was applied for
agarose gel electrophoresis.
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DISCUSSION |
The open reading frame of the DNase II cDNA (Fig. 9) can be
translated into a 364-amino acid polypeptide chain. As illustrated in
Fig. 11, a putative transmembrane
peptide at the NH2 terminus, two small connecting peptides
between
1 and
and between
and
2, and a peptide at the
COOH terminus are evidently removed from the nascent chain to form
mature DNase II. Thus, all three chains of DNase II are coded by the
same cDNA in the sequence
1,
, and
2. Removal of the
putative transmembrane peptide is a cotranslational event, but removal
of the other three small peptides probably occurs within lysosomes
after protein folding. This type of in vivo proteolytic
processing, quite common for lysosomal enzymes, has been described in
the maturation of cathepsin D (22), sialic acid
O-acetylesterase (23),
-mannosidase (24), and cysteine proteinase (25, 26).

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Fig. 11.
Schematic representation of the cDNA
coding region. The numbers are the amino acid residues
starting from Met, coded by the initiation codon. The shaded
areas are removed proteolytically to form mature DNase II.
Disulfide bridges are indicated as S-S . The triangles
represent the N-glycosylation sites.
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When the
and
subunits of DNase II are separated, the
polypeptides are unable to reconstitute to the active enzyme (12). However, whether the unprocessed polypeptide is active or not is
unknown. Thus, DNase II activity in the growth medium of transfected cells could be caused by one complete chain with no internal cleavages. As to where and when the addition and modification of carbohydrate side
chains occur, DNase II probably follows the same dogma of glycosylation
as do other lysosomal enzymes (27).
A regulation system for translocation of lysosomal enzymes has been
suggested (28), and in the absence of such a system many lysosomal
enzymes are secreted excessively into the extracellular compartment.
This regulation system normally maintains a constant concentration of
endogenous DNase II within lysosomes. However, when expression of DNase
II in transfected cells is turned on, apparently any excessive amount
of DNase II is secreted into the extracellular compartment, accounting
for the activities detected in the growth medium. In lower eukaryotes,
such as Tetrahymena, lysosomal enzymes are released into the
surrounding medium for nutritional purposes and are not merely a
consequence of exocytosis of secondary lysosomes (29). Whether the
presence of DNase II activity in the growth medium of transfected
Chinese hamster ovary cells as shown in Fig. 10 is a response to
intoxication or is part of an exocrine function remains to be
determined. However, it is possible that because of low transfection
efficiency (about 10%) only the transfected cells lysed, and as a
result their soluble intercellular contents including DNase II are
mixed with the growth medium.
The homology search shows that three human cDNA sequences (GenBank
accession numbers AA075967, H12842, and AA224257) are highly homologous
with the cDNA sequence of porcine spleen DNase
II.2 The functions of these
cDNA sequence-coding proteins are not known. Perhaps these
sequences are parts of the cDNA of human DNase II. Also, the
porcine DNase II cDNA sequence is similar to that of a cDNA of
Caenorhabditis elegans (GenBank accession number L11247)
encoding a protein of unknown function.
We thank Dr. Roger E. Koeppe of Oklahoma State
University for reading the manuscript.