DNase X is a glycosylphosphatidylinositol-anchored membrane enzyme that provides a barrier to endocytosis-mediated transfer of a foreign gene.

DNase X is the first mammalian DNase to be isolated that is homologous to DNase I. In this study, we have examined its function using a novel monoclonal antibody and showed it to be expressed on the cell surface as a glycosylphosphatidylinositolanchored membrane protein. High level expression was observed in human muscular tissues and in myotubes obtained in vitro from RD rhabdomyosarcoma cells. We observed that RD myotubes incorporated a foreign gene, lacZ, by endocytosis but that expression of the encoded coding product, beta-galactosidase, was strongly inhibited. Overexpression of DNase X inhibited endocytosis-mediated gene transfer, whereas knockdown of DNase X with small interfering RNA had the opposite effect. These results reveal that DNase X provides a cell surface barrier to endocytosis-mediated gene transfer.

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X.

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Identification of two functional nuclear localization signals in DNase gamma and their roles in its apoptotic DNase activity.

Among DNase I family members, only DNase gamma causes DNA fragmentation during apoptosis. However, the molecular basis for this functional feature of DNase gamma is poorly understood. Here we describe the identification of functional NLSs (nuclear localization signals) in DNase gamma and their roles in its apoptotic function. DNase gamma contains two NLSs: a classical bipartite-type NLS (NLS1) located in the N-terminal half, and a short basic domain (NLS2) at the C-terminus. No potential NLSs are found in the primary structures of other DNase I family DNases. Inactivation of either NLS1 or NLS2 causes reduced DNA ladder-producing activity in DNase gamma. Disruption of NLS2 suppresses ladder formation more effectively than disruption of NLS1. DNase gamma doubly mutated in both NLSs is enzymically active, but no longer catalyses apoptotic DNA fragmentation. Although DNase I fails to produce ladder formation during apoptosis, DNase I fused to NLS2 of DNase gamma through its C-terminus is able to catalyse DNA fragmentation in apoptotic cells. These results indicate that the presence of either NLS1 or NLS2 is necessary for the apoptotic function of DNase gamma, and that the most important domain for this function is NLS2. These findings also explain the lack of apoptotic DNase activity in the other DNase I family DNases.