Knock down of cytosolic phospholipase A2: an antisense oligonucleotide having a nuclear localization binds a C-terminal motif of glyceraldehyde-3-phosphate dehydrogenase.
We have previously shown that an antisense, effective in the knock down of cytosolic phospholipase A2 (cPLA2), localizes mainly in the nucleus of human endothelial cells and monocytes and that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is involved in its nuclear localization. In this study, we clarify how GAPDH participates in the nuclear localization of this antisense oligodeoxynucleotide (ODN) directed against cPLA2 mRNA. A central TAAAT motif providing specificity and high affinity binding was assumed to interact with the enzyme Rossmann fold region on the basis of competition to this site by NAD+. To asses whether the TAAAT motif interacts directly with the enzyme Rossmann fold region, we evaluated the binding to GAPDH of different oligonucleotides and the effect of competitors such as NAD+, NADH, mononucleotides, DNA, polyribonucleic acids and polyanions. We found that the dissociation constant for TAAAT containing oligonucleotides was three--to fivefold higher with respect to oligo not containing this motif. By covalently linking 32P-labeled cPLA2p(N)16 to GAPDH and after executing hydrolysis with hydroxylamine, the labeling was exclusively found in the C-terminal domain (aa 286-334). These results indicate that the antisense oligonucleotide interacts with a site not having a defined function but which can be negatively allosterically regulated when NAD+ or polynucleotides are bound to Rossmann fold.
Laktionov
Pavel
P
Institute of Bioorganic Chemistry, Academy of Sciences Siberian Division, Novosibirsk, Russia.
Rykova
Elena
E
Toni
Mattia
M
Spisni
Enzo
E
Griffoni
Cristiana
C
Bryksin
Anton
A
Volodko
Natalia
N
Vlassov
Valentin
V
Tomasi
Vittorio
V
D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000378:metabolism*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2
D016376:Oligonucleotides, Antisense; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010741:Phospholipases A; D054467:Phospholipases A2