Myristoyl-CoA:protein N-Myristoyltransferases: Isoformidentification and gene expression in retina

[摘要] Purpose: The mammalian myristoyl-CoA:protein N-myristoyltransferase(NMT) family consists of Type I and Type II enzymes that typicallycatalyze the addition of myristic acid (14:0) to the N-terminus ofspecific proteins using myristoyl-Coenzyme A as a donor. However, theN-terminus of certain proteins in frog and bovine retina are modifiedwith fatty acids other than myristic acid, and this utilization ofalternative acyl-CoAs has not been found in any other tissues. Thisalternative use of acyl groups is known as retina heterogeneousacylation and occurs by an unknown mechanism. Therefore, the focus ofthis work has been to identify NMT isoforms that may be unique ordifferentially expressed in bovine retina that may utilize acyl-CoAs inaddition to myristoyl-CoA.Methods: We used cDNA library screening, RT-PCR, and PCR to identifyNMTs in bovine retina and liver, then compared these proteins topreviously described NMTs by amino acid alignments. We used northernblotting to determine the level of Type I and Type II NMTs in a humanmulti-tissue blot, in bovine retina, and in bovine liver. Southernblotting was done to determine if one or more genomic copies of Type IINMT are present in the bovine genome. A phylogenetic analysis of NMTisoforms is provided to describe the lineage of NMTs from a variety ofspecies.Results: We identified a Type I NMT in retina that is nearlyidentical to previously described Type I enzymes, as well as a novelType II enzyme. We further characterized the retina specificity of thenovel enzyme by evaluating which Type II enzymes are present in bovineliver. We find that cow liver contains a Type II NMT identical to thepreviously described NMT found in human liver, as well as a partialclone that is identical to the novel Type II NMT from retina. NMT Type Iand II message expression appears to vary in a panel of tissues, and wesuggest that retina expresses at least three NMT isoforms, with the TypeI short form and Type II being more abundant than the Type I long formNMT. We also present evidence that there are two copies of Type II NMTin the bovine genome.Conclusions: Our efforts to find a retina-specific NMT resulted inthe identification of a previously unknown Type II NMT, but this enzymealso appears to be expressed in liver. Therefore, we are unable toconclude that heterogeneous acylation occurs by means of an exclusiveenzyme capable of efficiently utilizing multiple acyl-CoA substratessolely in retina. Nonetheless, our results lead to an improvedunderstanding of protein acylating enzymes and encourage us to furtheridentify factors that influence tissue-specific expression of eachisoform and regulate their enzymatic activity in terms of acyl-CoAselectivity and heterogeneous protein acylation.

[学科分类] 生物化学/生物物理