Porcine Spleen Deoxyribonuclease II

Abstract

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked ␣⅐␤ heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708 -10713). The ␣ subunit, after disulfide cleavage, yields two chains, ␣1 and ␣2. The complete amino acid sequences of the ␣1, ␤, and ␣2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between ␣1 and ␣2 and of three intrachain disulfides in ␣2 were assigned. Six carbohydrate attachment sites, two in ␤ and four in ␣2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH 2 -end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence ␣1, ␤, and ␣2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.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 3Ј-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 ␣⅐␤ 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 PROCEDURESMaterials-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 T...

Biogenesis and Proteolytic Processing of Lysosomal DNase II

“…There are two hypotheses regarding maturation of the DNase II protein. Biochemical purification of porcine DNase II indicated that the enzyme is composed of two or more (α1, α2 and β) subunits [13], [22], [23], [37]. However, it has been argued that these subunits are artificially generated during purification and the protein is active as a single polypeptide.…”

“…The proposed α2 subunit of porcine splenic DNase II, which is the fragment closest to the carboxyl-terminal, starts from Ser110 and ends with Lys351 [23]. In murine DNase II, the amino acid residues that correspond to these sites are Ser110 and Glu351 of mouse DNase II, respectively.…”

“…Purified porcine DNase II was determined by gel filtration to have a molecular weight of 45 kDa, but SDS-PAGE showed molecular weights of 35 and 10 kDa [22]. Although processing of porcine DNase II by proteases has been proposed [23], [24], human DNase II does not seem to undergo processing [18], [19].…”

The Genome of Fowlpox Virus

“…The DNase II homologue, FPV032, is truncated compared to the previously described FPV gene, FPCEL-1 (93). FPV032 represents the largest subunit (␣2) of cellular DNase II and includes the conserved histidine at the potential active site (99,174). The function of this gene in the viral replication cycle is unknown; however, FPCEL-1 is not Continued on facing page essential for viral growth in vitro (93).…”

Structure of acid deoxyribonuclease

“…Although Bth DNase had not been enzymatically characterized previously, both pig DNase II (43–45) and human DNase IIα (24,46,47) have been characterized. Mutational analyses have shown that H113 and H295 of human DNase IIα are essential for activity, but H295 mutant enzymes could still interact with DNA (24).…”