Uptake, intracellular distribution, and novel binding proteins of immunostimulatory CpG oligodeoxynucleotides in microglial cells.
Microglial cells are central components of the innate immune system of the brain and contribute to inflammatory and degenerative processes. DNA with unmethylated CpG dinucleotides is a potent stimulant of microglial cells. We have analyzed uptake, intracellular distribution, and cellular binding proteins of CpG oligdeoxynucleotides (ODNs) by the microglial cell line N9. The uptake of CpG-ODN is concentration-, time-, and temperature-dependent, but, interestingly, independent of the CpG dinucleotides. After internalisation, CpG-ODN localized to the cytoplasm and showed a typical speckled distribution pattern. We further purified the cellular binding proteins of CpG-ODN and identified several binding proteins by tryptic digestion and mass spectrometry. Most of the CpG-ODN binding proteins are RNA processing enzymes, which are important for RNA splicing, export, and stability. Further, we identified a protein, pigpen, which has not been observed in microglial cells, so far. These proteins apparently bind CpG-ODN with low selectivity, as binding is independent of CpG dinucleotides. Interference of immunostimulatory and therapeutic oligonucleotides with proteins and enzymes of RNA transport and processing has not been described so far and might affect the physiological functions of these proteins and also might influence cellular localization of therapeutic ODN. These findings are helpful in understanding the cellular fate of ODN and the nonsequence-specific effects of ODN and for rational design and evaluation of ODN-based therapeutic strategies.
Zhang
Zhiren
Z
Institute of Brain Research, University of Tuebingen, Calwer Street 3, Tuebingen D-72076, Germany. zhangzhiren@yahoo.com
Weinschenk
Toni
T
Schluesener
Hermann J
HJ
D036002:ADP Ribose Transferases / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D018899:CpG Islands / Q000276:immunology; D004247:DNA / Q000378:metabolism; D016264:Dextran Sulfate / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000276:immunology* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000096:biosynthesis; D009838:Oligodeoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011766:Pyruvate Carboxylase / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D002352:Carrier Proteins; C423449:CpG ODN 1826; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides; D013873:Thionucleotides; D009569:Nitric Oxide; D004247:DNA; D016264:Dextran Sulfate; C007789:fucoidan; D036002:ADP Ribose Transferases; D011766:Pyruvate Carboxylase; D016650:Fluorescein-5-isothiocyanate