Myristoyl-CoA:protein N-myristoyltransferases: isoform identification and gene expression in retina

The mammalian myristoyl-CoA:protein N-myristoyltransferase (NMT) family consists of Type I and Type II enzymes that typically catalyze the addition of myristic acid (14:0) to the N-terminus of specific proteins using myristoyl-Coenzyme A as a donor. However, the N-terminus of certain proteins in frog and bovine retina are modified with fatty acids other than myristic acid, and this utilization of alternative acyl-CoAs has not been found in any other tissues. This alternative use of acyl groups is known as retina heterogeneous acylation and occurs by an unknown mechanism. Therefore, the focus of this work has been to identify NMT isoforms that may be unique or differentially expressed in bovine retina that may utilize acyl-CoAs in addition to myristoyl-CoA.

We used cDNA library screening, RT-PCR, and PCR to identify NMTs in bovine retina and liver, then compared these proteins to previously described NMTs by amino acid alignments. We used northern blotting to determine the level of Type I and Type II NMTs in a human multi-tissue blot, in bovine retina, and in bovine liver. Southern blotting was done to determine if one or more genomic copies of Type II NMT are present in the bovine genome. A phylogenetic analysis of NMT isoforms is provided to describe the lineage of NMTs from a variety of species.

We identified a Type I NMT in retina that is nearly identical to previously described Type I enzymes, as well as a novel Type II enzyme. We further characterized the retina specificity of the novel enzyme by evaluating which Type II enzymes are present in bovine liver. We find that cow liver contains a Type II NMT identical to the previously described NMT found in human liver, as well as a partial clone that is identical to the novel Type II NMT from retina. NMT Type I and II message expression appears to vary in a panel of tissues, and we suggest that retina expresses at least three NMT isoforms, with the Type I short form and Type II being more abundant than the Type I long form NMT. We also present evidence that there are two copies of Type II NMT in the bovine genome.

Our efforts to find a retina-specific NMT resulted in the identification of a previously unknown Type II NMT, but this enzyme also appears to be expressed in liver. Therefore, we are unable to conclude that heterogeneous acylation occurs by means of an exclusive enzyme capable of efficiently utilizing multiple acyl-CoA substrates solely in retina. Nonetheless, our results lead to an improved understanding of protein acylating enzymes and encourage us to further identify factors that influence tissue-specific expression of each isoform and regulate their enzymatic activity in terms of acyl-CoA selectivity and heterogeneous protein acylation.