Galactosylated histone-mediated gene transfer and expression.
We have developed a novel, highly efficient DNA delivery system to accomplish gene transfer through the asialoglycoprotein receptor-mediated endocytosis pathway. Natural nuclear DNA-binding proteins, the histones (H1, H2a, H2b, H3, and H4), were modified and used as receptor-targeted DNA carriers. Galactosylated with a coupling agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the histones and albumin were conjugated to DNA and then used to transfect HepG2 cells, which display the asialoglycoprotein receptor. The extent of galactosylation was determined for all histone subgroups and albumin with 14C-labeled galactose. A reporter gene for the bacterial chloramphenicol acetyltransferase (CAT), under the control of the 5' long terminal repeat (LTR) of Rous sarcoma virus, was used for comparisons of transfection efficiency of various carrier proteins. The CAT activity resulting from histone H1-mediated transfection was 1.66 unit per 10(6) cells, the highest among histone subgroups. The galactyosylated histone H1 was also eleven times more effective than the asialo-orosomucoid-polylysine. Ten galactosyl units are attached to histone H1 by the galactosylation reaction. Differences in the extent of galactosylation could not explain different transfection efficiencies among various proteins studied in this report. Treatment with galactose oxidase abolished the transfection ability of both the galactosylated histone H1 and asialo-orosomucoid. The intrinsic DNA-binding domains and nuclear location signal sequences are unique to histones as receptor-targeted DNA carriers, and are advantageous for effective gene delivery.
Chen
J
J
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
Stickles
R J
RJ
Daichendt
K A
KA
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004274:DNA, Recombinant / Q000008:administration & dosage*; D004705:Endocytosis; D005690:Galactose / Q000494:pharmacology; D005692:Galactose Oxidase / Q000494:pharmacology; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006031:Glycosylation; D006657:Histones / Q000737:chemistry*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D009961:Orosomucoid / Q000031:analogs & derivatives; D011107:Polylysine; D011956:Receptors, Cell Surface / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D006657:Histones; D009961:Orosomucoid; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C017030:asialoorosomucoid; D011107:Polylysine; D005692:Galactose Oxidase; D015500:Chloramphenicol O-Acetyltransferase; D005690:Galactose