Porcine Spleen Deoxyribonuclease II

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked azb heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708–10713). The a subunit, after disulfide cleavage, yields two chains, a1 and a2. The complete amino acid sequences of the a1, b, and a2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between a1 and a2 and of three intrachain disulfides in a2 were assigned. Six carbohydrate attachment sites, two in b and four in a2, 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 NH2-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 a1, b, and a2 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.