|Molecular Vision 2004;
Received 20 December 2003 | Accepted 16 February 2004 | Published 22 March 2004
Myristoyl-CoA:protein N-Myristoyltransferases: Isoform identification and gene expression in retina
Dana R. Rundle,1 Raju V. S. Rajala,1,2,3 Richard A.
Alvarez,4 Robert E.
Departments of 1Cell Biology, 2Ophthalmology, and 4Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 3Dean McGee Eye Institute, Oklahoma City, OK
Correspondence to: Dr. Robert E. Anderson, Dean McGee Eye Institute, 608 S. L. Young Boulevard, Room 409, Oklahoma City, OK, 73104; Phone: (405) 271-8250; FAX: (405) 271-8128; email: firstname.lastname@example.org
Purpose: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
The co-translational transfer of the myristoyl group from myristoyl-CoA to the N-terminus of over one hundred proteins by N-myristoyltransferase (NMT) enzymes facilitates protein-protein interactions , targets proteins to the membrane , and is necessary for normal growth and development of the Arabidopsis thalania plant , for proper viral maturation , and for vegetative growth of several pathologic fungi [5-7]. The NMT family is composed of Type I and Type II enzymes that are 77% homologous, with the N-terminus being the region of lowest similarity between the two types . The Type I NMT group consists of a long and a short form of the enzyme. The short form starts at an internal methionine in the long form and lacks a putative poly-lysine ribosomal targeting signal (KKKKKKQKRKKEK) found at the 5' end of long forms in both Type I and II enzymes [9,10]. Type II enzymes have been previously identified in mouse and human liver, and their most obvious difference is a 31 amino acid region present in the N-terminus of mouse Type II NMT that is not present in the human Type II enzyme . There is not a clear understanding for the necessity of highly homologous enzymes having a presumably redundant function in vivo, but differences in subcellular localization and peptide substrate preferences may be possible reasons for the presence of multiple NMT enzymes [11-15].
Heterogeneous acylation is a retina-specific phenomenon in which transducin, recoverin, guanylate cyclase activating protein (GCAP), and the cAMP csatalytic dependent kinase A subunit (CDK) are modified with myristic acid (14:0), tetradecenoic acid (14:1n-9), tetradecadienoic acid (14:2n-6), and lauric acid (12:0) [16-19]. In vitro studies of the heterogeneously acylated proteins have suggested that the varying hydrophobicities of the acyl groups can provide slightly different binding affinities to the acylated protein, and these differences could generate interaction gradients with the membrane or other proteins to attenuate the visual signal [16,20-22]. A previous study concluded that protein heterogeneous acylation does not occur due to higher levels of 14:1n-9-CoA, 14:2n-6-CoA, and 12:0-CoA than 14:0-CoA in retina because the pool sizes of these molecules in retina are not significantly different from their pool sizes in bovine heart and liver . In support of this, if acyl-CoA pool size were the determining factor, then retina proteins would each be modified to the same extent with each acyl group. Instead, bovine retina proteins vary significantly in their degree of modification with each acyl group .
Because a mechanism for retina heterogeneous acylation has not been adequately explained by analysis of acyl-CoA pools and NMT enzymes have not been characterized in retina, our goal has been to examine NMT in bovine retina for the possibility of a unique, retina-specific enzyme. In this study we show that there are multiple NMTs expressed in retina, one of which is a novel Type II enzyme that is the third member of the Type II category. We have also compared NMT mRNA expression levels in bovine and human tissues, identified Type II enzymes in bovine liver to assess the tissue specificity of the novel enzyme found in retina, and used Southern blotting to evaluate the bovine genome for Type II NMT gene copy number.
NMT identification by PCR
PCR was performed using retina cDNA (Stratagene, La Jolla, CA) as a template and gene-specific primers (5'-ATGAACTCTTTGCCAGCAGAGAG-3' and 5'-TCTGCTCCCCTTGCCAGG-3') based on the bovine spleen Type I NMT sequence . Additional 5' nucleotide sequence for the Type I NMT was obtained by PCR using bovine retina cDNA provided by Dr. Wolfgang Baehr (University of Utah, Salt Lake City, UT) using 5'-TGGCGGAC/TGAGAGTGAGACA-3' and 5'-TCGAACCCCACAGTGCCAC-3' primers. Bovine liver QUICK Clone cDNA (Clontech, San Jose, CA) was PCR amplified with primers corresponding to the nucleotide sequences of amino acids 1-6 and 493-499 of the human liver Type II NMT using Platinum Taq polymerase (Gibco/BRL, Gaithersburg, MD). PCR products were analyzed on a 1% agarose/TAE gel and candidate bands were isolated from the gel for reamplification. PCR products were sequenced with the above primers and identified by BLAST search.
Retina and liver cDNA library screening
A bovine retina cDNA library (Stratagene; approximately 1.0x106 plaques) was screened according to standard screening protocols . Pre-hybridization was with 2X PIPES buffer, 50% deionized formamide, 0.5% SDS, and 100 μg/ml sonicated herring sperm DNA for 8 h at 42 °C. A 1.3 kb 32P radiolabeled probe (RadPrime, Gibco/BRL) encompassing the coding region of a human liver Type I NMT was used to screen duplicate filter lifts at 42 °C overnight, followed by autoradiography. Nineteen clones were sequenced with vector-specific primers and found to be Type I NMT by BLAST analysis. Further sequencing was performed using gene-specific primers to cover the full length of the inserts. The bovine retina library (approximately 1.5x106 plaques) was also screened for Type II NMT as described above using a 1.5 kb 32P labeled probe amplified by PCR from human liver Type II NMT. Five Type II NMT positive clones were identified by BLAST analysis after sequencing.
Approximately 1x106 plaques from a bovine liver cDNA library (Stratagene) were screened for Type II NMT. Pre-hybridization was in ULTRAhyb (Ambion, Austin, TX) for 8 h and probed overnight at 42 °C using the 1.5 kb 32P labeled Type II probe from Type II human liver NMT. The filters were washed twice in 0.1% SSC with 0.1% SDS at room temperature, and twice at 55 °C prior to autoradiography. Six clones were sequenced with T3 and T7 primers and found to be Type II NMT by BLAST analysis. Further sequencing was performed using gene-specific primers to cover the full length of the clones.
The complete 5' end of the retina NMT Type II 5' coding sequence was generated by reverse transcription of 5 μg bovine retina total RNA isolated from fresh tissue with TriReagent (Sigma, St. Louis, MO) using Thermoscript (Gibco/BRL) at 63 °C with a gene-specific primer (GSP-B: 5'-GGCGAGTAATCAAACCGGAAC-3') according to Gibco/BRL protocol. The reverse transcription product was used for PCR amplification with gene-specific primers (5'-ATGGCGGAGGACAGCGAGTCT-3') and GSP-B using Platinum Taq High Fidelity polymerase (Gibco/BRL). The PCR product was visualized on a gel, excised, inserted into pT7 Blue vector using a Perfectly Blunt Cloning kit (Novagen. Madison, WI), and transformed into SURE cells (Stratagene). This insert was sequenced and identified by BLAST search.
RNA isolation and northern blotting
Fresh bovine retinas and liver were obtained from the Oklahoma State University College of Veterinary Medicine, Stillwater, OK. Total RNA was isolated from this tissue with TriReagent (Sigma) according to the manufacturer's protocol. The RNA (10 μg per lane) was fractionated on a 1.0% formaldehyde denaturing agarose gel, vacuum transferred to BrightStar membrane (Ambion) for one hour in 20X SSC according to standard procedures, then UV crosslinked. Pre-hybridization and hybridization were done in ULTRAhyb at 42 °C and probed with 32P labeled DNA (1.0x106 cpm/ml) corresponding to the complete coding region of Type I or Type II NMT. The membrane was washed twice in 2X SSC containing 0.1% SDS, followed by two washes at 42 °C in 0.2X SSC containing 0.1% SDS and a single wash at 55 °C in 0.1X SSC containing 0.1% SDS, followed by autoradiograph. A human multi-tissue northern blot (Clontech, 2 μg mRNA per lane) was similarly probed for Type I and II NMTs.
Bovine genomic DNA (8 μg per digestion) was restriction enzyme digested and fragments were separated on a 0.8% agarose gel in TBE by standard protocols. The gel was transferred to Hybond XL (Amersham Pharmacia Biotech, Piscataway, NJ) membrane in 20X SSC, prehybridized in ULTRAhyb, followed by hybridization overnight at 42 °C with a 32P radiolabeled cDNA probe (1.0x106 cpm/ml) corresponding to nucleotides 1-819 of the retina Type II enzyme. The membrane was processed as described for northern blotting.
Results & Discussion
Enzyme identification and analysis
Our first approach to identify NMT enzymes in retina was to PCR amplify bovine retina cDNA using primers based on the first and final eighteen nucleotides of the coding sequence reported for bovine spleen Type I NMT . Because this resulted in amplification of a product identical to the bovine spleen enzyme, a larger effort was subsequently undertaken to identify any other Type I NMT that might exist in bovine retina by cDNA library screening. Library screening resulted in nineteen NMT Type I positive clones and each clone was sequenced to identify possible homologues or splice variants. All nineteen clones represented a single sequence, but none contained the entire coding sequence described for the long form of human liver Type I enzyme , although one clone contained all but the first 12 amino acids of this sequence. Therefore, it was probable that a similar long form N-terminal sequence existed for bovine retina Type I NMT, but was not well represented in the cDNA library used for screening. The 5' region of Type I NMT could be difficult to amplify due to a poly-A region (86% dATP, 31 of 36 nucleotides) followed by a region containing two sequences 60 and 68% GC-rich over 30 and 55 nucleotide spans, respectively. The loose conformation of the poly-A region followed by the more tightly constrained GC rich regions could cause the reverse transcriptase to stall, leading to a population of cDNA truncated at the 5' end. A second source of retina cDNA was obtained and successfully used as a PCR template to generate a product containing the complete 5' sequences as well as regions that overlapped and completely matched the sequence of Type I found by retina cDNA library screening. The complete Type I NMT sequence was deposited in Genbank (accession number AF223384). A comparison of Type I NMT amino acid sequences from human liver and muscle (hNMT I), human hepatocarcinoma HepG2 cells (short form, hNMTS I HepG2), bovine retina (bNMT I retina), bovine spleen and bovine retina (short form, NMTS I sp/ret), and mouse liver (mNMT I) is shown in Figure 1. The Type I NMTs from human sources do not vary in amino acid sequence except for initiation at an internal site of the long form to generate the short form, as found in HepG2 cells. The long form of bovine retina Type I NMT differs from human and mouse liver at the second amino acid where the alanine found in human and mouse enzymes is replaced by glycine in the bovine enzyme. Additionally, the bovine retina full-length enzyme (bNMT I) has an asparagine at position 494 that is not found in any other Type I NMT. Overall, Type I NMTs from cow, mouse, and human have a high level of identity, with human and cow being most closely related.
The bovine retina cDNA library was screened for Type II NMT, which resulted in five positive clones that were a single sequence highly homologous to human and mouse liver Type II NMT , but lacking an initiating methionine and the N-terminal poly-lysine region present in other Type II NMTs. The human liver NMT Type II N-terminal coding sequence also has poly-A and G-C rich regions and the five retina Type II NMT clones were truncated just 3' of the poly-A region, indicating that structural conflicts similar to those of Type I NMT could have also generated N-terminally truncated Type II NMT the retina cDNA library. The complete 5' end of the retina Type II enzyme was generated using a high temperature reverse transcriptase to amplify new bovine cDNA from RNA isolated from fresh retina. The RT-PCR product served as the template for PCR amplification using gene specific primers. This PCR product was subcloned into a vector for sequencing and provided the complete 5' coding sequence for bovine retina Type II NMT. The retina Type II NMT represents a third, unique member of the Type II enzyme family and has been deposited in Genbank (accession number AF222687).
To determine whether the novel retina Type II enzyme was a retina-specific enzyme potentially responsible for retina protein heterogeneous acylation or a species-specific form of the enzyme present in other bovine tissues, PCR amplification of bovine liver cDNA using primers to regions conserved in all known Type II enzymes was used to amplify Type II NMTs from bovine liver. PCR generated a single product that was identical to human liver NMT Type II, but lacked the 3' UTR. Because the 3' UTR appears to be the region of least homology among NMTs (Figure 2) and could provide insight into regulatory differences between the enzymes found in bovine liver and bovine retina, a bovine liver cDNA library was screened to obtain additional 3' UTR sequence. Six bovine liver Type II clones were sequenced and found to be identical to bovine retina Type II NMT starting at amino acid 54 (bNMT II retina, Figure 3), but lacking a complete 5' end including a starting methionine. This was an unexpected finding since PCR of bovine liver cDNA had previously generated a single enzyme identical to human liver Type II NMT, not to bovine retina Type II NMT. Possible reasons for the presence of both NMT Type II isoforms could be that multiple NMT Type II enzymes are normally present in liver, the cDNAs used for PCR and library screening were from different sources and could represent developmental differences in Type II NMT expression in liver, or represent the preferential expression of an isoform by unknown factors capable of regulating NMT expression in liver. However, this is the first evidence that multiple Type II NMTs are present in a single tissue type. We were unable to compare 3'UTRs because we were ultimately unable to generate a match to the bovine Type II found by subsequent PCR amplification or screening. Liver Type II NMT sequence data from PCR and library screening were deposited in Genbank (accession numberAF232826 and AY007993, respectively). The amino acid sequences of Type II NMT from bovine retina and liver are aligned with mouse and human liver Type II NMTs in Figure 3. However, the bovine liver Type II NMT (bNMT1 II liver) complete N-terminal amino acid sequence not found in the library or amplified by PCR could differ from that of the bovine retina Type II NMT (bNMT II retina) sequence. That possible result would then generate both bovine retina and liver specific Type II NMT enzymes. Given the high level of identity at the N-termini of three known Type II enzymes shown in Figure 3, and that bovine retina (bNMT) and liver (bNMT1) Type II NMTs are identical throughout the remainder of the protein, makes it likely that the complete 5 ends will be identical as well.
The human liver Type II NMT (hNMT II liver) has a potential internal starting methionine (Met 88), but this amino acid has been replaced by a valine in the bovine retina Type II MNT and by isoleucine in mouse Type II NMT. Although both human and bovine Type II NMT have a methionine at amino acid position 98, this is not presumed to be an initiating methionine since human liver Type II expression in COS-7 cells did not produce a truncated Type II NMT . Therefore, unlike the Type I NMTs, there is no evidence to date that the Type II enzymes have an internal initiating methionine or that they generate short and long forms of the enzyme. The consequence of the 31 additional amino acids in mouse liver Type II NMT is not known, but this region would not affect heterogeneous acylation since that does not occur in liver and because enzymes in retina where heterogeneous acylation does occur do not have a similar N-terminal region. The otherwise relatively minor amino acid differences between human, mouse, and cow may reflect species based variations. A phylogenetic analysis (Figure 4) of NMTs from mammals, insects, fungi, and plants indicates that the bovine retina Type II (bNMT II retina) segregates with mouse liver Type II (mNMT II liver), whereas the long form of bovine retina Type I NMT (bNMT I retina) segregates with human liver Type I NMT (hNMT I liver).
Northern blot analysis
A northern blot of bovine retina and liver total RNA was probed for NMT Type I, NMT Type II, and actin (Figure 5). Using actin expression as an internal control, Type II NMT had greater expression in bovine retina than in bovine liver. Although liver may have multiple NMT TypeII isoforms, only a single 4.2 kb band was observed. Using a Type I probe, 2.8 kb and 5.6 kb bands were detected in bovine retina and bovine liver, and the overall expression of both Type I NMT messages was less in bovine retina than in bovine liver. Bovine liver showed nearly equal expression of the 2.8 kb and 5.6 kb messages, while retina indicated a differential expression with the shorter message being the predominant form. This is consistent with the retina library screening results in that 11 of the 19 clones began at or just 5' of the internal initiating methionine (Met 88), while several others were truncated at or near a second internal methionine (Met 110). A human multi-tissue northern blot containing 2 μg of heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas mRNA was probed for actin, Type I NMT, and Type II NMT (Figure 6). Two bands of comparable size to that seen for bovine Type I NMT were present in all of the human tissues represented on the blot. Heart, skeletal muscle, and liver showed a high level of short Type I message expression, whereas brain and kidney showed a predominance of the long Type I message. Human heart, brain, skeletal muscle, kidney, pancreas, and liver express Type II NMT, but placenta and lung contained very little of this isoform. The results indicate that the Type I short form has a higher expression level than the full length Type I enzyme in bovine retina, as well as being the predominant message in five of eight human tissues represented on the multi-tissue blot. The expression of Type II NMT in bovine retina is approximately four times the Type II NMT expression level in bovine liver. Assuming that human liver and bovine liver have comparable levels of Type II NMT expression, and that bovine retina has higher Type II expression than bovine liver, it is reasonable to suggest that retina could be a very high NMT Type II expressing tissue.
It has been reported that there are two genomic copies for bovine Type I NMT , although sequence differences among known Type I forms are minor and the shorter form appears to originate by initiation at an internal methionine . Because the Type II NMTs identified in cattle have sequence differences that cannot be readily explained by alternative splicing or alternative internal initiation sites, a Southern blot analysis for Type II NMT was done to assess the number of Type II NMT genomic copies in the cow genome (Figure 7). Genomic DNA was restriction enzyme digested with Bam HI, Hind III, or Bam HI and Hind III together, followed with Southern blotting and hybridization to a Type II probe corresponding to the first 273 amino acids of human Type II NMT. This region has the least homology to Type I NMT and did not react with Type I NMT plasmid DNA used as a control (data not shown). The single restriction enzyme digestions generated bands different than those reported for a Type I NMT Southern blot . The double restriction enzyme digestion produced a 2 kb band that hybridized to the Type II probe that was not present in the single digests. This band could represent a second genomic copy having a restriction enzyme site not present in the other copy. This result taken together with the nucleotide differences in the two bovine Type II enzymes suggests that two genomic copies may exist for the Type II enzyme and this would explain the presence of two Type II isoforms in bovine liver.
In conclusion, we have identified both Type I and Type II NMTs in bovine retina. We have identified a novel Type II NMT isoform in bovine retina, although rigorous efforts to identify other retinal Type II NMTs by PCR and library screening were not successful. Type I NMT in retina exists as a long form containing a putative ribosomal targeting sequence, and as an N-terminally truncated enzyme lacking this targeting sequence. Northern blot indicates long and short Type I NMT and Type II mRNAs are present in retina with the short Type I and Type II NMTs being the more abundant enzymes. However, a partial clone of the Type II NMT identified in retina was also found in bovine liver and a sequence identical to human liver Type II NMT was also PCR amplified from bovine liver. This would indicate that liver expresses more than one form of Type II enzyme and that NMTs are regulated in a tissue specific manner. Southern blotting suggests that the presence of two forms of Type II NMT in cow liver results from the presence of two genomic copies. This result is supported by the fact that the sequence differences found in the these Type II enzymes could not originate from alternative splicing of a single transcript because the amino acid changes are random but frequently in close proximity to each other. Evaluation of NMT message expression by northern blotting indicates that Type I and II enzymes are differentially regulated in a tissue-specific manner, and we further propose that retina is a high Type II NMT expressing tissue in comparison to liver.
The high degree of identity between human liver and bovine retina Type II NMTs and the high probability that the retina Type II form may also exist in liver, makes it difficult to conclude that retinal heterogeneous acylation occurs from a retina-specific NMT having unique structural or enzymatic characteristics. In vitro kinetic studies with the recombinant NMTs cloned in this study have exhibited catalytic activity with four acyl-CoAs known to acylate retinal proteins in vivo . It has been shown that heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulate raft localization and signal transduction . This study also demonstrated that modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its level of raft localization and resulted in decreased T cell signal transduction . Recent studies have shown that transducin, a heterogeneously acylated protein, is associated with lipid rafts in bovine retinal rod outer segments . These studies imply that heterogeneous acylation may be capable of regulating signal transduction by membrane-bound proteins and raises the interesting possibility that transducin, an important component of the visual transduction pathway, may be sequestered in different domains of ROS membranes depending on the nature of the N-terminal fatty acid. We further suggest that heterogeneous acylation is not be a unique function of a single NMT, but it may be dependent on the subcellular localization of the enzyme or upon factors that can either regulate the tissue specific expression or catalytic specificity of a variety of NMT isoforms.
We would like to thank Dr. Ben Cravatt of the Scripps Institute for his generous gift of human and mouse liver Type I and Type II NMT plasmids and Holly Whiteside for technical assistance in preparing this manuscript. This work was supported by grants from the National Institutes of Health/National Eye Institute (EY00871, EY04149, and EY12190) and from Research to Prevent Blindness Inc., New York, NY; The Foundation Fighting Blindness, Baltimore, MD; Samuel Roberts Nobel Foundation, Inc., Ardmore, OK; and Presbyterian Health Foundation, Oklahoma City, OK.
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