INTRODUCTION

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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.

	EXPERIMENTAL PROCEDURES

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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.

	RESULTS

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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.

View larger version (19K): [in this window] [in a new window]   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.

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.

View larger version (25K): [in this window] [in a new window]   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).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (27K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   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.

View larger version (28K): [in this window] [in a new window]   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.

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.

View larger version (22K): [in this window] [in a new window]   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).

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).

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 II71-76), and a 3' degenerate primer, 5'-AGRTCRTCNCCRAARTTNCC-3' (R = A or G; antisense; DNase II145-150). 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 II100-106), and a 3'-specific primer, 5'-GGGCCAGGAAAGGCTATCTGGG-3' (antisense; DNase II170-177), 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).

View larger version (6K): [in this window] [in a new window]   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.

View larger version (48K): [in this window] [in a new window]   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.

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.

View larger version (17K): [in this window] [in a new window]   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.

	DISCUSSION

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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).

View larger version (7K): [in this window] [in a new window]   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.

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.