MUTAGENESIS OF APOBEC-1, THE CATALYTIC SUBUNIT OF THE MAMMALIAN APOLIPOPROTEIN-B MESSENGER-RNA EDITING ENZYME, REVEALS DISTINCT DOMAINS THAT MEDIATE CYTOSINE NUCLEOSIDE DEAMINASE, RNA-BINDING, AND RNA EDITING ACTIVITY

Apolipoprotein (apo) B48 is synthesized by mammalian small intestine a s a result of post-transcriptional RNA editing. This process is mediat ed by an enzyme complex containing a catalytic subunit, apobec-1, whic h is homologous to other cytidine deaminases, particularly in a domain (H/C)-(A/V)-E-(X)(24-30)-P-C-(X)(2)-C which coordinates zinc. apobec- 1, expressed as a glutathione S-transferase fusion protein, demonstrat es both apoB RNA editing and cytidine deaminase activity. His(61) Cys( 93), and Cys(96), the putative zinc-coordinating residues, were mutate d to Arg, Ser, and Ser, respectively, with loss of RNA editing activit y and either great reduction or abolition of cytidine deaminase activi ty. Mutation of the catalytically active Glu(63) residue to Gln and pr o(92) to Leu abolished both cytidine deaminase and RNA editing activit y. The conservative His(61) --> Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zin c binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repea t of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity, GS T/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation o f His(61) --> Arg abolished RNA binding, while the Glu(63), Gln and Cy s(96) --> Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression o f wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His(61) --> Cys, L RR, and Cys(93) --> Ser mutants increased endogenous editing 23-fold, while Glu(63) --> Gln and His(61) --> Arg mutants acted as dominant ne gatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the contex t of the apoB mRNA editing enzyme.