Proteolytic processing of porcine deoxyribonuclease II occurs in lysosomes but is not required for enzyme activation.
DNase II purified from porcine spleen (pDNase II) comprises alpha(1), alpha(2) and beta subunits. The three subunits are encoded by one cDNA, in the sequence alpha(1), beta, and alpha(2), and the peptides linking these subunits are presumably cleaved out post-translationally. To understand the relevance of post-translational cleavage to pDNase II, recombinant pDNase II (rpDNase II) was produced in human 293T cells by transfection with an expression plasmid containing pDNase II cDNA (pcDNaseII). An 11.5, a 35 and a 46.5 kDa protein were detected in the cell lysates, whereas only a 46.5 kDa protein was detected in the culture medium of the pcDNaseII-transfected cells. The 46.5 kDa rpDNase II secreted into the medium was purified to homogeneity and characterized. MALDI-TOF MS and N-terminal amino acid sequencing of the 46.5 kDa protein revealed a single contiguous polypeptide chain of pDNase II. Zymographic analysis showed that the 46.5 kDa protein digested DNA in acidic conditions and that the specific activity of this rpDNase II was about twice that of pDNase II purified from porcine spleen. Treatment with chloroquine, a lysosomal inhibitor, resulted in the accumulation of only the 46.5 kDa protein in the pcDNaseII-transfected cells. Treatments with cycloheximide 22 h after transfection led to accumulation of the processed enzyme and disappearance of the 46.5 kDa protein. These results suggest that the proteolytic processing of rpDNase II occurs in the lysosome, which is not involved in the activation of pDNase II.
Huang
Ru-Ting
RT
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Liao
Ta-Hsiu
TH
Lu
Shao-Chun
SC
D000818:Animals; D002478:Cells, Cultured; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II