Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression.
Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, 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 alpha1, beta, and alpha2 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.
Wang
C C
CC
Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lu
S C
SC
Chen
H L
HL
Liao
T H
TH
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013154:Spleen / Q000201:enzymology*; D013552:Swine
D018076:DNA, Complementary; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II