Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase

Molecular and Cellular Biochemistry 189: 91–97, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

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Recombinant bovine spleen myristoyl CoA: Protein N-myristoyltransferase Rajala V.S. Raju,1 Raju S.S. Datla,2 Rakesh Kakkar1 and Rajendra K. Sharma1 1

Department of Pathology and Saskatoon Cancer Centre, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan; 2Plant Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan, Canada Received 26 November 1997; accepted 1 April 1998

Abstract Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the co-translational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. Recently, we have isolated full length cDNA encoding bovine spleen NMT [27] the full length cDNA was cloned and expressed in E. coli, resulting in the expression of functionally active 50 kDa NMT. Using the combination of SP-Sepharose fast flow and Mono S fast protein liquid chromatography, the enzyme was purified 20-fold with a high yield. The spleen NMT (sNMT) fusion protein exhibited an apparent molecular weight of 53 kDa on SDS-PAGE. Upon cleavage by the Enterokinase the sNMT exhibited an apparent molecular weight of 50 kDa without loss of catalytic activity. The two synthetic peptide substrates based on the N-terminal sequence of pp60src (GSSKSKMR) and cAMP dependent protein kinase (GNAAAKKRR) have different kinetic parameters of Km values of 40 and 200 µM. Recombinant sNMT was also potently inhibited by Ni2+ (histidine binder) in a concentration dependent manner with a half maximal inhibition of 280 µM. The E. coli expressed sNMT was homogenous and showed enzyme activity. (Mol Cell Biochem 189: 91–97, 1998) Key words: myristoyltransferase, myristoyl-CoA, lipid modification, purication, expression Abbreviations: NMT – N-myristoyl-CoA:protein N-myristoyltrasferase; hNMT – human NMT; sNMT – spleen NMT; SDS – sodium dodecyl sulfate; FPLC – fast protein liquid chromatography; IPTG – isopropyl β-D-thiogalactopyranoside; ARF – ADP-ribosylation factor

Introduction Myristoyl CoA-protein N-myristoyltransferase (NMT) catalyses the co-translational transfer of myristate from myristoyl CoA to the amino terminal glycine residue of a number of cellular, viral and oncoproteins (see reviews [1–3]. These proteins include, the catalytic subunit of cAMPdependent protein kinase, various tyrosine kinases (including pp60src, pp60yes, pp56lck, pp59fyn/syn, and cAbl), the β subunit of calmodulin-dependent protein phosphatase (calcineurin), the myristoylated alanine rich C kinase substrate, the α subunit

of several G proteins, and several ARF proteins involved in ADP ribosylation [1–5]. In addition, many viruses have been shown to have myristoylated coat proteins, and myristoylation of these proteins has been demonstrated to be important for their replication [6]. Heteroatom-substituted analogs of the myristoyl group and inhibitors of NMT have been reported to inhibit the replication of a number of viruses, including HIV [7]. Fungi also myristoylate a number of proteins which are important to the viability of these organisms [8]. Studies with Candida albicans and Crypotcoccus neoformans have

Address for offprints: R.K. Sharma, Department of Pathology and Saskatoon Cancer Centre, 209, 20 Campus Drive, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 4H4, Canada

92 also shown that myristoylation is important for the vegetative growth of these pathogens [8]. The incorporation of myristoyl group analogs into the fungal proteins was reported to result in cell death [8]. Saccharomyces cerevisiae strain with a mutant allele (nmt 181) exhibits global defects in protein myristoylation and undergoes growth arrest at various stages of the cell cycle [9]. We have demonstrated, for the first time, elevated levels of NMT activity in animal and human colon cancer tissues suggesting a role for NMT in tumor progression [10]. Recently we have also demonstrated the overexpression of NMT protein in colorectal adenocarcinomas [11]. The fact that both viral, fungal and tumor growth can be inhibited by perturbation of myristoylation suggest that the enzyme responsible for this protein modification could provide a suitable target for therapeutic agents. The purpose of choosing to isolate and study bovine spleen NMT (sNMT) was that the oncogene pp60 src has been reported to be abundant quantities in spleen [12]. The importance of myristoylation of proteins in tumorigenesis was suggested by studies demonstrating that myristoylation of viral oncogene product pp60v-src, is required for membrane association and cell transformation [13–15]. Blockage of the myristoylation of pp60 c-src in colonic cell lines depressed colony formation, cell proliferation, and localization of pp60 c-src to the plasma membrane [16]. The function and subcellular localization of other proteins involved in the signal transduction are variously altered by mutations resulting in loss of myristoylation [1–3, 17, 18]. Therefore, there is evidence to support the hypothesis that myristoylation of proteins may be involved in the pathogenesis of the cancer. Myristoylation has been identified as a possible approach to the development of chemotherapeutic agents [19, 20]. Synthetic inhibitors of NMT activity have been developed on the basis of analogs of the substrates and products of NMT (acyl-CoA), peptide and the acyl peptide [3, 7]. An endogenous inhibitor of NMT purified from bovine brain [21] inhibited NMT activity in the rat colonic tumors in vitro [10]. The availability of specific inhibitors of this enzyme will facilitate further study of its role in the development of cancer. In order rationally to design selective inhibitors of NMT, a more detailed characterization of mammalian enzyme is needed. Since this depends on being able to obtain sufficient quantities of the protein, we sought to overcome the difficulties of purification from tissues. Therefore we have expressed this enzyme in E. coli and purifying the recombinant protein by sequential chromatographic steps. Also described kinetic analysis of recombinant sNMT.

Materials and methods Materials [1-14C]myristoyl-CoA (54.7 mCi/mmol) was obtained from Amersham. pTrcHisC vector was obtained from Invitrogen. SP-Sepharose fast flow matrix and Mono S HR 5/5 FPLC column were purchased from Pharmacia Biotechnologies Inc. Agarose Gel DNA extraction kit was purchased from Boehringer Mannheim, Canada. Pseudomonas acyl CoA synthetase, coenzyme A, benzamidine, phenylmethylsulfonyl fluoride and leupeptin were obtained from Sigma. General laboratory chemicals were of analytical grade.

Peptide synthesis The following peptides were synthesized by the solid phase manual protocol developed by Merrifield [22] Gly-Asn-Ala-Ala-Ala-Ala-Lys-Lys-Arg-Arg (based on the NH 2 terminal sequence of type II catalytic subunit of cAMP-dependent protein kinase) and Gly-Ser-Ser-Lys-SerLys-Pro-Lys-Arg (the NH2 terminal sequence of pp60src). The peptides were purified by CM-Cellulose column chromatography and G-25 Sephadex gel filtration.

Assay of N-myristoyltransferase Myristoyltransferase activity was measured as described earlier [23, 24]. For the standard enzyme assay, the reaction mixture contained 50 µM radiolabelled myristoyl-CoA, 50 mM Tris-HCl, pH 7.4, 0.5 mM EGTA, 1% Triton X-100, peptide and NMT in a total volume of 25 µl. The transferase reaction was initiated by the addition of radiolabelled myristoyl-CoA and was incubated at 30°C for 10–30 min. The reaction was terminated by spotting aliquots of incubation mixture onto P81 phosphocellulose paper discs and drying them under a stream of warm air. The P81 phosphocellulose paper discs were washed in three changes of 40 mM Tris-HCl, pH 7.3 for 90 min. The radioactivity was quantified in 7.5 ml of Beckman Ready Safe Liquid Scintillation mixture, in a Beckman Liquid Scintillation Counter. One unit of NMT activity was expressed as 1 nmol of myristoyl peptide formed per min.

Other methods SDS-Polyacrylamide gel electrophoresis was carried out according to the procedure of Laemmli [25] employing 10% gels. Coomassie Brilliant Blue was used to visualize

93 the protein bands on the gel. Protein concentration was determined by the method of Bradford [26] using bovine serum albumin as a standard.

Results Construction of E. coli expression vector The bovine sNMT cDNA [27] which contains the entire coding sequence except the first 8 amino acids at the N-terminal end was isolated from the pBluescript SK (+) vector by digesting the plasmid with BamHI and EcoRI and separating the fragment on 0.8% agarose gel. The cDNA insert was purified using Agarose Gel DNA Extraction Kit (Boehringer Mannheim, Canada). The fragment was ligated into pTrcHisC which had been digested with BamHI and EcoRI. The derivative recombinant plasmid was designated as pTrcHisC.sNMT (Fig. 1A). The resulting fusion protein generated by this construct had a poly-histidine followed by 24 amino acids that contains the Enterokinase cleavage site and the NMT protein from its ninth amino acid (Fig. 1B).

Expression and purification of recombinant sNMT E. coli DH5α with the recombinant pTrcHisC.sNMT was grown to stationary phase at 37°C in LB medium containing 50 mg/L ampicillin and 1 mM final concentration of isopropyl β-D-thiogalactopyranoside. The bacteria were harvested by centrifugation at 10,000 × g for 20 min. The bacteria were suspended in buffer A [50 mM Tris-HCl, pH 8.0 containing, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin and 0.1 % Triton X-100]. The bacterial suspension was sonicated for 10 sec on ice. The lysate was cleared by centrifugation at 15,000 × g for 20 min, and the pellet was discarded.

Mono S HR (5/5) FPLC chromatography Dialyzed SP-Sepharose pooled sample was loaded onto the Mono S column preequlibrated with buffer B using a 50 ml Pharmacia superloop. The column was developed at a flow rate of 1 ml/min with buffer B. The column was washed with 5 bed volumes of buffer B. The NMT activity was eluted by a salt gradient in buffer B from 0–400 mM NaCl in 40 min followed by washing with 1.0 M NaCl. The NMT activity was eluted as a sharp peak between 280 and 300 mM NaCl (Fig. 2A). Fractions having NMT activity were pooled, and SDS-polyacrylamide gel electrophoresis was carried out to examine its purity (Fig. 2B). The pooled samples were stored in aliquots at –70°C until use.

Purification, purity and molecular weight Table 1 summarizes the data of a typical preparation of the enzyme from approximately 1 litre of E. coli culture. The recombinant sNMT was usually purified between 20–25-fold from E. coli lysates with 38% recovery. The Mono S FPLC yielded 2.0 mg of protein with a specific activity of 48 nmol/ min/mg in the presence of pp60src derived peptide substrate. The Mono S FPLC fraction showed a single band of 53.8 kDa with an apparent homogeneity on SDS PAGE (Fig. 2B). The sNMT fusion protein at the N-terminal end contains poly-histidine followed by an Enterokinase cleavage site [Asp4-Lys]. Upon cleavage of the fusion protein product by the Enterokinase, the molecular weight of sNMT was found to be 50 kDa (data not shown). The fusion protein has a poly-histidine tag which has a high affinity for divalent cations such as nickel and could permit one step purification of recombinant protein from crude lysates. However, purification of sNMT by Ni2+ affinity chromatography resulted in a significant loss of enzyme activity (data not shown). Further, Ni2+ was also found to inhibit the sNMT activity in a concentration dependent manner with a half maximal inhibition of 280 µM (Fig. 3).

SP-Sepharose fast flow column Bacterial lysate was applied to a SP-Sepharose fast flow column (8.5 × 20 cm) which was preequlibrated with buffer B [50 mM potassium phosphate buffer, pH 7.0, 0.1 mM EGTA and 10 mM 2-mercaptoethanol]. After application, the column was washed with 2 bed volumes of buffer B. The column was further washed with 1.5 column volumes of buffer B containing 75 mM NaCl until no protein was detected by a dye binding method [26]. The bound protein was eluted with buffer B containing 0.3 M NaCl. Fractions containing high NMT activity were pooled and dialysed over night against buffer B.

Table 1. Purification of recombinant bovine sNMT

Fraction Lysate SP Sepharose fast flow Chromatography Mono S FPLC chromatography

Total activity unit

Total Specific Purification Yield protein activity -fold mg unit/mg

240

113

150

15

10

96

2

48

2.2

1.0

100

4.5

63

21

38

One unit of NMT activity is defined as 1 nmol of myristoylated peptide formed per min.

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A

Fig. 1. Schematic representation of the constructed plasmid pTrcHisC.sNMT. (A) The BamHI to EcoRI fragment was cloned into pTrcHisC which encode the entire open reading frame for the sNMT except the first 8 amino acids. Predicted N-terminal sequence of the expressed sNMT; (B) The sequence indicated the enterokinase cleavage site [Asp4-Lys] with dotted line. The sNMT lacks residues 1–8 as the internal 5′ BamHI site in the sNMT gene [27] was used in the construction of pTrcHisC.sNMT.

Kinetic parameters of recombinant sNMT

5′ untranslated sequence of bovine spleen NMT

Kinetic properties of the purified recombinant sNMT were carried out using two synthetic peptides derived from the N-terminal sequences of pp60 src and cAMP-dependent protein kinase. The results suggest that NMT has a higher Vmax, towards to pp60src as compared with cAMP-dependent protein kinase. Recombinant spleen NMT had 5 fold lower Km for pp60 src (40 µM) as compared with cAMP-dependent protein kinase (200 µM) peptide substrate.

Figure 4 shows that 5′ untranslated sequence (ULS) of sNMT contains a polylysine block similar to the sequence reported previously on human and bovine brain NMT [28].

Discussion The recombinant sNMT enzyme was expressed in E. coli DH5α cells with transcription driven by trc promoter. The functional protein was expressed as a poly-histidine tagged

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Fig. 2. (A) Mono S FPLC chromatography. Pooled NMT activity from SP Sepharose fast flow column was applied on to Mono, S FPLC column as described in Results section. Ten microliters of samples of the elute were assayed for NMT activity. The major NMT activity peak was eluted between 280 and 300 mM NaCl; (B) SDS-Polyacrylamide gel electrophoresis of recombinant sNMT. Gel electrophoresis was carried out with 20 µg of purified sNMT. Molecular weight markers used in this study were: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and trypsin inhibitor (20.1 kDa).

fusion protein with an N-terminal extension of 34 aa. The pTrcHisC vector has a poly-histidine tag followed by an Enterokinase cleavage site. Upon cleavage of the fusion protein product, the molecular weight of sNMT was found to be 50 kDa, similar to the molecular weight reported for the NMT purified from the bovine spleen tissues [29]. In addition, the Enterokinase cleavage did not effect the catalytic activity of the enzyme, suggesting that the fusion protein activity was not due to the extra amino acids at the N-terminal end. Previously we have expressed hNMT from a full length cDNA utilizing both a T7 RNA polymerase gene expression system [30] and as a poly-histidine tagged form [31]. The two expressed proteins were similar in their specific activities suggesting the N-terminal poly-histidine tag had no influence on the catalytic activity of the enzyme. Recombinant sNMT was purified to near homogeneity employing a cation exchange chromatography on a SP-Sepharose fast flow and Mono S FPLC. The procedure is rapid, simple, and reproducible and suitable for large scale preparation. The purified enzyme has an apparent molecular weight of 53.8 kDa on SDS PAGE (Fig. 2B). The enzyme exhibited a specific activity of 48 nmol/min/mg protein in the presence of pp60 src derived peptide substrate. This is similar to the activity reported for the purified enzyme from the bovine spleen tissues [29]. The enzyme was purified more than 20-fold from E. coli lysates with greater than a 35% yield. The fusion protein has a poly-histidine tag, which has a high affinity for divalent cations such as nickel, and could permit one step purification of recombinant protein from the crude lysates. Purification by Ni 2+ Sepharose chromatography resulted in the loss of sNMT activity (data not shown). Previous studies on nickel column chromatography for the purification of hNMT has been shown to result in a significant loss of enzyme yield [32]. In addition, Ni 2+ was found to inhibit the sNMT activity in a concentration dependent manner with a half maximal inhibition of 280 µM. The loss of sNMT activity is due to the blocking of L-histidine residues which are important for the catalytic activity, as has been shown previously [32–34]. The recombinant sNMT has a low Km for pp60src (40 µM) than for cAMP-dependent protein kinase derived peptide substrate (200 µM). Similar values were reported for the spleen enzyme purified previously from the bovine spleen tissues [29]. Expression and availability of wild type or mutant sNMT could provide a powerful system to study the underlying mechanism of regulation of many myristoylated proteins in the mammalian cells. The NH2-terminal domain of yeast [35], human [36], bovine [27, 37] and Drosophila NMT [38] are not required for in vitro catalytic activity. Recently it has been shown that human NMT amino-terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction [28]. Further authors have also reported the NH2-terminal 60 kDa hNMT

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Fig. 3. Concentration dependent inhibition of bovine sNMT activity by Ni2+. NMT (3.0 µg) was incubated with Ni2+ (0.039–2.5 mM) in the presence of cAMP dependent protein kinase derived peptide, substrate (1.0 mM) and carried out NMT assay at 30°C for 10 min as described under Materials and methods. Results are expressed as percent of control (NMT activity in the absence of Ni2+). Assays were done in duplicate.

contains a polylysine block encompassing amino acids 37– 50 (KKKKKKQKKKKEKG) with a high homology to basic stretches found in human and rat N-methionylaminopeptidases, eIF-2β subunit of the eukaryotic initiation factor, human eIF-2β and yeast eIF-2β [28]. Similar polylysine stretch is also found in the 5′ ULS of bovine spleen NMT. The 5′ untranslated sequence of bovine spleen NMT is identical to the sequenced reported for bovine brain and human NMT [28, 39]. Previously we have reported that bovine NMT is likely encoded by two copies of NMT gene [27] and no information is available on the genomic DNA whether these two copies are functional. Further characterization of genomic DNA would give better information on the functionality of these 2 copies. Analysis of the sequence between the junction of the assigned initiation codon (Fig. 4) ATG is preceded by nucleotide AG [AG-

ATG]. Most of the introns follow dinucleotide rule, terminates with AG [40]. It could be possible the different molecular size NMTs reported from different tissues could be by an alternative RNA splicing mechanism i.e. in larger molecular masses this intron could be a part of the coding sequence whereas the lower molecular mass NMT could be spliced products. Such a possibility can not be ruled out.

Acknowledgments Dr. R.V.S. Raju is a recipient of a Research Fellowship from the Health Services Utilization and Research Commission of Saskatchewan.

Fig. 4. 5′ untranslated sequence of bovine spleen NMT. Untranslated sequence was translated into protein . Sequence taken from Raju et al. [27]. [AG-ATG], junction before the assigned initiation codon methionine (int).

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