Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylat ion sites (N-86, N-212, and N-266) Of human deoxyribonuclease II (DNase II) , conserved in mammalian enzymes, and a proteolytic processing site (Q(46)- R-47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N-86 and N-266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II act ivities, irrespective of cell. line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneou s attachment of a carbohydrate moiety to N-86 and N-266, cleavage of the pr opeptide from the single DNase II precursor, and the inherent signal peptid e might be required for subcellular sorting and proteolytic maturation of t he enzyme. (C) 1999 Academic Press.