Revised structure of the active form of human deoxyribonuclease IIalpha.
Deoxyribonuclease IIalpha (DNase IIalpha) is an acid endonuclease found in lysosomes, nuclei, and various secretions. Murine DNase IIalpha is required for digesting the DNA of apoptotic cells after phagocytosis and for correct development and viability. DNase IIalpha purified from porcine spleen was previously shown to contain three peptides, two of which were thiol crosslinked, all derived by processing of a single polypeptide. Commercial bovine protein is consistent with this structure. However, screening of 18 human cell lines failed to demonstrate this processing, rather a 45 kDa protein was consistently observed. Incubation of cells with the N-glycosylation inhibitor tunicamycin resulted in a 37 kDa protein, which is close to the predicted formula weight. The protein also contains at least one thiol crosslink. Similar results were obtained with overexpressed DNase IIalpha. These results suggest that active DNase IIalpha consists of one contiguous polypeptide. We suggest the previous structure reflects proteolysis during protein purification.
MacLea
Kyle S
KS
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Krieser
Ronald J
RJ
Eastman
Alan
A
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006031:Glycosylation; D006801:Humans; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D013438:Sulfhydryl Compounds; D014415:Tunicamycin; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II; D003545:Cysteine