Reconstitution of TCP80/NF90 translation inhibition activity in insect cells

Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, a n inherited metabolic prototype for enzyme and gene therapy. An 80-kDa mamm alian cytoplasmic protein (TCP80/NF90) was discovered to interact with the GCase mRNA coding region and inhibit its translation in vitro and ex vivo. Human TCP80/NF90 is identical to NF90, an IL-2 enhancer protein, and MPP4, an M-phase phosphoprotein. The interaction of recombinant TCP80/ NF90 with GCase mRNA was evaluated using the baculovirus/Sf9 insect cell system since these cells lack this protein. Purified recombinant and isolated mammalian cytoplasmic TCP80/NF90 had identical functions including binding of coding regions of selected RNAs and inhibition of their in vitro translation. Ind ividual baculoviruses containing the human TCP80/NF90 cDNA (vSf9/CP80) and GCase cDNA (vSf9/GCase) were used to co-infect Sf9 cells. The presence of p reformed TCP80/NF90 significantly (>87%) inhibited wild-type GCase mRNA tra nslation in these cells, but baculovirus containing a mutant GCase did not. Sf9 cells co-infected with vSf9/TCP80 showed a major reduction of GCase RN A polysome association. These results show that the multifunctional protein , TCP80/NF90, can function in vivo as a translation inhibitory protein and include alterations of mRNA binding to polysomes as a component of its mech anism of action. (C) 2000 Academic Press.