MacGinnitie A J, Anant S, Davidson N O
Department of Medicine, University of Chicago, Illinois 60637, USA.
J Biol Chem. 1995 Jun 16;270(24):14768-75.
Apolipoprotein (apo) B48 is synthesized by mammalian small intestine as a result of post-transcriptional RNA editing. This process is mediated by an enzyme complex containing a catalytic subunit, apobec-1, which 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, demonstrates both apoB RNA editing and cytidine deaminase activity. His61, Cys93, and Cys96, the putative zinc-coordinating residues, were mutated to Arg, Ser, and Ser, respectively, with loss of RNA editing activity and either great reduction or abolition of cytidine deaminase activity. Mutation of the catalytically active Glu63 residue to Gln and Pro92 to Leu abolished both cytidine deaminase and RNA editing activity. The conservative His61-->Cys mutation, which should coordinate zinc, retained both editing and cytidine deaminase activity. Thus, zinc binding is required for both apoB RNA editing and cytidine deaminase activity. Mutation of the first four leucines within the heptad repeat of the leucine-rich region (LRR) of apobec-1 resulted in reduced RNA editing but preservation of wild-type cytidine deaminase activity. GST/APOBEC-1 was also demonstrated to cross-link to apoB RNA. Mutation of His61-->Arg abolished RNA binding, while the Glu63-->Gln and Cys96-->Ser mutant proteins showed wild-type levels of RNA binding. The remaining mutants had reduced levels of activity. Overexpression of wild-type apobec-1 in McA 7777 cells resulted in a 5-6-fold increase in editing of endogenous apoB. Transfection of the His61-->Cys, LRR, and Cys93-->Ser mutants increased endogenous editing 2-3-fold, while Glu63-->Gln and His61-->Arg mutants acted as dominant negatives, reducing endogenous editing. These data suggest that apobec-1 has distinct functional domains which modulate activity in the context of the apoB mRNA editing enzyme.
载脂蛋白（apo）B48是哺乳动物小肠通过转录后RNA编辑合成的。这一过程由一种含有催化亚基载脂蛋白B mRNA编辑酶1（apobec-1）的酶复合物介导，apobec-1与其他胞苷脱氨酶同源，特别是在一个与锌配位的结构域（H/C）-（A/V）-E-（X）24-30-P-C-（X）2-C中。以谷胱甘肽S-转移酶融合蛋白形式表达的apobec-1，兼具载脂蛋白B RNA编辑和胞苷脱氨酶活性。假定的锌配位残基组氨酸61（His61）、半胱氨酸93（Cys93）和半胱氨酸96（Cys96）分别突变为精氨酸（Arg）、丝氨酸（Ser）和丝氨酸，导致RNA编辑活性丧失，胞苷脱氨酶活性大幅降低或完全丧失。将具有催化活性的谷氨酸63（Glu63）残基突变为谷氨酰胺（Gln）以及脯氨酸92（Pro92）突变为亮氨酸（Leu），会使胞苷脱氨酶和RNA编辑活性均丧失。保守的组氨酸61突变为半胱氨酸（His61-->Cys），本应与锌配位，却保留了编辑和胞苷脱氨酶活性。因此，锌结合对于载脂蛋白B RNA编辑和胞苷脱氨酶活性均是必需的。apobec-1富含亮氨酸区域（LRR）七肽重复序列中的前四个亮氨酸发生突变，导致RNA编辑减少，但野生型胞苷脱氨酶活性得以保留。谷胱甘肽S-转移酶/载脂蛋白B mRNA编辑酶1（GST/APOBEC-1）也被证明能与载脂蛋白B RNA交联。组氨酸61突变为精氨酸（His61-->Arg）会使RNA结合丧失，而谷氨酸63突变为谷氨酰胺（Glu63-->Gln）和半胱氨酸96突变为丝氨酸（Cys96-->Ser）的突变蛋白显示出野生型水平的RNA结合。其余突变体的活性水平降低。在McA 7777细胞中过表达野生型apobec-1，导致内源性载脂蛋白B的编辑增加5至6倍。转染组氨酸61突变为半胱氨酸（His61-->Cys）、富含亮氨酸区域（LRR）和半胱氨酸93突变为丝氨酸（Cys93-->Ser）的突变体，使内源性编辑增加2至3倍，而谷氨酸63突变为谷氨酰胺（Glu63-->Gln）和组氨酸61突变为精氨酸（His61-->Arg）的突变体则起显性负作用，降低内源性编辑。这些数据表明，apobec-1具有不同的功能结构域，在载脂蛋白B mRNA编辑酶的背景下调节活性。
