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Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115
Reprint requests to: You-Di Liao, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115; e-mail: ydliao{at}ibms.sinica.edu.tw; fax: 886-2-27829142.
(RECEIVED February 5, 2004; FINAL REVISION April 1, 2004; ACCEPTED April 1, 2004)
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Keywords: N-terminal Met; E. coli MetAP; substrate specificity; penultimate residue; ribonuclease
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04679104.
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Introduction |
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Recently, methionine aminopeptidases (MetAPs) have been used to remove the N-terminal Met in Escherichia coli and yeast. There are two types of MetAP, MetAP I and MetAP II, respectively, in eukaryotes. Only MetAP I exists in eubacteria and only MetAP II exists in archaea (Li and Chang 1995; Tahirov et al. 1998). These MetAP genes are essential for the growth of prokaryotes and eukaryotes (Chang et al. 1989; Li and Chang 1995). However, the efficiency of Met removal is limited by the side-chain radius (<3.68 Å) of the penultimate residue, the second residue after Met (Ben-Bassat et al. 1987; Hirel et al. 1989; Hwang et al. 1999; Chen et al. 2002). From the structure of E. coli MetAP and bestatin-based inhibitor complex, it has been discovered that four residues (Tyr 168, Gln 233, Met 206, and Glu 204) reside in the substrate-binding pocket (Fig. 1
from PDB 3MAT
[PDB]
; Lowther et al. 1999; Lowther and Matthews 2000). The Met 329 and Gln 356 residues of yeast MetAP I, corresponding to Met 206 and Gln 233 of E. coli MetAP, have been replaced with Ala. With an oligopeptide as the in vitro substrate, the purified MetAP I variants (M329A, Q356A) exhibit a significant increase in catalytic activities for oligopeptides with slightly larger penultimate residues, such as Asn, His, and Met, but not for those with bulky and acidic residues (Roderick and Matthews 1993; Walker and Bradshaw 1999).
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, lane 2). The yields of MetAP varied with the type of coexpressed target protein (Fig. 2A, lanes 5 and 7), for example, 50 mg purified protein was obtained per 1 L of culture in the absence of target protein. The yield of coexpressed target proteins varied with the property of the protein, for example, the amount of frog ribonucleases (onconase and RC-RNaseL1) is different as shown in Fig. 2B, lanes 6 and 8).
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) was replaced by a small residue, for example, Gly, Thr, Asp, or Asn, and coexpressed with the onconase that begins with MQD. The percentage of Met removal was calculated by dividing the amount of proteins initiated with the penultimate residue by the amount of total proteins (i.e., proteins initiated with the penultimate residue plus unprocessed protein initiated with Met) after Edman degradation. The Met preceding Gln was removable by MetAP-Q233G (74% of Met removal) and MetAP-Q233T (88%). Furthermore, a second residue, Met 206, in the pocket (Fig. 1) was replaced by a small residue, for example, Gly, Thr, or Val. The Met of MQD-Onc was also removable by MetAP-M206G (85% of Met removal), MetAP-M206T (86%), and MetAP-M206V (61%). To make the MetAP more competent, we made a double mutation at residues Met 206 and Gln 233. For the substrate MQD-Onc, the double mutants, MetAP-M206G-Q233G (96%), MetAP-M206T-Q233G (91%), MetAP-M206T-Q233T (93%), and MetAP-M206V-Q233T (90%) variants, designated as MetAP-*GG, MetAP-*TG, MetAP-*TT, and MetAP-*VT, respectively, possess high in vivo catalytic activities (Table 1; data not shown). For other substrates, only MetAP-*TG exerted high catalytic activity for MLD-Onc (86% Met removal) and MID-Onc (22%; Table 1), whereas the MetAP-*GG did not, regardless of the apparent large pocket for substrate binding (data not shown).
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) was further replaced with a small residue, Gly, Ser, Thr, Val, Asp, or Glu. For the substrate MID-Onc, the efficiency of Met removal by the parental MetAP-*TG was 22%, but it increased markedly to 94% by Y168G mutation and to 68% by Y168T mutation, which were designated as MetAP-GTG and MetAP-TTG, respectively (Table 1). In contrast, for substrates with an acidic penultimate residue, for example, MDD-Onc or MED-Onc, the third Y168T mutation exhibited higher activity than that of the Y168G mutation (Table 1). The result suggests that the hydroxyl group of residue 168 of E. coli MetAP with a short side chain (Thr) may play a crucial role in the recognition of the acidic penultimate residue.
The substrate specificity of engineered MetAPs
The substrate specificities of MetAP variants were determined by using MXD-Onc as a substrate that possesses a different penultimate residue (Table 1). The Met preceding the moderate penultimate residue—that is, Asn (85%), Leu (86%), His (92%), Gln (90%), and Met (100%)—as well as a small and uncharged residue—that is, Gly (95%), Ala (99%), Pro (93%), Ser (99%), Thr (85%), and Val (80%)—were mostly removed by the MetAP-*TG. For proteins with a bulky penultimate residue, Ile and Trp, most of Met was removed by MetAP-GTG, at 94% and 78%, respectively. For protein with acidic penultimate residues, Asp and Glu, the Met was also removable by MetAP-TTG, at 80% and 58%, respectively. However, the Met preceding bulky basic penultimate residues, Lys and Arg, is still not removable by these variants.
Effect of antepenultimate residue on substrate specificity
Using MXD-Onc as a substrate, we demonstrated the importance of the penultimate residue on the substrate specificity of engineered MetAP in vivo (Table 1). However, we also found that the substrate specificity of MetAP was influenced by the antepenultimate residue. With MAX-Onc as a substrate, the removal of terminal Met was not significantly altered by the change of the antepenultimate residue, whereas both terminal Met and penultimate Ala were removable by the engineered MetAPs when the antepenultimate residue was small, that is, Gly or Ala (Table 2). With MLX-Onc or MQX-Onc as a substrate, the terminal Met and penultimate Leu/Gln residues were also removable by engineered MetAPs when the antepenultimate residue was small or moderate, such as Gly, Ala, Ser, Thr, Val, or Asn, but not bulky or charged, such as Asp, Glu, Phe, Trp, Lys, or Arg. Furthermore, both Met and Phe/Trp residues were removable by MetAP-GTG when the antepenultimate residue was Ala but not Asp (Tables 1, 2). With respect to wild-type MetAP, the N-terminal Met and penultimate Ala of MAA- or MAG-initiated onconase and RC-RNase 4 were also removable (Tables 2, 3).
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). This result suggests that the Pro residue at the antepenultimate site may exert a steric hindrance on the catalytic activity of the MetAP enzyme.
Applications in other recombinant proteins
To investigate the possible application of engineered MetAPs in the production of recombinant proteins, we used several soluble or insoluble proteins as substrates. The Met of insoluble MQD-initiated bullfrog ribonucleases (RC-RNase 3, RC-RNase 4, RC-RNase 6, and RC-RNase L1-N2D) was removed by MetAP-*TG, and both Met and Gln of insoluble MQN-initiated bullfrog ribonucleases (RC-RNase 2 and RC-RNase L1) were removed by the enzyme (Table 3). The terminal Met of MQD-initiated glutathione S-transferase (GST) of Schistosoma japonicum, either soluble or insoluble, was also removable by the MetAP-*TG. Both Met and Leu of the soluble MLA-initiated WW domain of CA150 protein, a transcription elongation regulator, were also removable (Table 3). To increase the competence of the engineered MetAP, we cloned the MetAP-*TG gene in the vector pET29b, which possesses kanamycin-resistance, and we cloned the gene of target protein, for example, onconase, the WW domain of CA150, or GST, in a separate expression vector pET22b, which possesses ampicillin resistance and is coexpressed in E. coli BL21(DE3). We found that the Met of these MQD- or MLD-initiated proteins was also removable, but with a slightly lower efficiency (10%–15% less; Table 3). This reduction may be due to lower copy number of the plasmid and the RNA transcript encoding the engineered MetAP.
From the eukaryotic curated (NP) protein sequence entries of the NCBI RefSeq database (dated January 7, 2003) using the SignalP program, we found that 37.4% of 5298 secretory proteins possesses a small residue, such as Gly, Ala, Cys, Pro, Ser, Thr, or Val, at N termini and 58.5% of 17,978 nonsecretory proteins possesses a small penultimate residue. The N-terminal Met of these recombinant proteins may be processed by wild-type MetAP. On the basis of the results we obtained by the coexpressed MetAPs, up to 84.5% of secretory proteins and 90.2% of nonsecretory proteins should be produced in authentic form.
Properties of recombinant ribonucleases
N terminus
The N terminus of MetAP-processed MQD-initiated on-conase was Gln, determined by Edman degradation, but it was a mixture of Gln/Pyr soon after refolding from inclusion bodies determined by mass spectrum analysis. One value (11,818 daltons) agreed with the mass of Pyr-initiated onconase (secretory or native onconase) and the other (11,835 daltons) agreed with that of Gln-initiated onconase calculated by the ExPASY server from the DNA sequence (Wilkins et al. 1998). The reduction of 17 daltons indicates that Pyr is derived from Gln through deamination. Similar results were also observed in the recombinant RC-RNase L1 proteins.
Catalytic activity
The catalytic activity of onconase was markedly reduced by the addition of Met and disruption of Pyr at the N terminus (100-fold less). The catalytic activity of onconase (rm-Onc) was restored after Met removal and refolding, similar to that of secretory or native onconase assayed by both zymogram (Fig. 2B, left panel) and the acid-insoluble method (data not shown). The catalytic activity of demethi-oninylated and refolded rmRCL1-N2D beginning with MQD was similar to that of ribonuclease isolated from bullfrog liver (RCL1) or secretory ribonuclease purified from culture medium (sRCL1), but that of MetAP-processed wild-type ribonuclease (rmRCL1) beginning with MQN was not fully restored (Fig. 2B, right panel), probably due to the partial deletion of terminal Gln. These results reveal that the removal of N-terminal Met by engineered MetAP is crucial for the catalytic activity of MQD-initiated frog ribonuclease.
In vitro catalytic activities of MetAP variants
For oligopeptides ranging from tetra- to dodecapeptides with Ala at the penultimate site, the wild-type MetAP and MetAP-*TG possessed high catalytic activity, whereas the MetAP-*GG, MetAP-*TT, and MetAP-GTG variants did not (Table 4). For the oligopeptide with Gln at the penultimate residue, wild-type and most engineered MetAPs did not exhibit significant catalytic activities, whereas the MetAP-GTG exerted 43% catalytic activity for the dodeca-peptide MQDWLTFQKKHI in comparison with that for MADWLTFQKKHI, which is identical to the N-terminal 
-helix of rM-Onc. The low in vitro catalytic activity of the purified MetAP for MQD-initiated oligopeptides suggests that some factor(s) may be required for the in vivo removal of Met from a bulky penultimate residue.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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In addition to the penultimate residue, the antepenultimate residue of a substrate may affect MetAP activity. The catalytic activity of human or yeast MetAP was reduced when the penultimate Val/Thr residue was followed by a Pro, for example, the MVP-initiated human 
-globin (Prchal et al. 1986) or MVP- or MTP-initiated yeast iso-1-cytochrome c (Moerschell et al. 1990). In contrast, both terminal Met and penultimate Ala residues were removed by E. coli MetAP when the antepenultimate residue was Pro, for example, MAP-initiated human interleukin-2 (Ben-Bassat et al. 1987). Thus, other aminopeptidase(s) could be responsible for the removal of the penultimate Ala residue in vivo (Ben-Bassat et al. 1987). Our results showed that the N-terminal Met and penultimate Ala/Leu/Gln/Phe/Trp residue were removed in vivo by engineered MetAP when the antepenultimate residue was small. The terminal Met and penultimate Ala of MAA-initiated onconase or RC-RNase 4 were also removed by the wild-type E. coli MetAP (Tables 2, 3). Once the terminal Met is removed by a MetAP variant, the small antepenultimate residue may serve as a penultimate residue and enable the subsequent cleavage of a new terminal residue of the demethioninylated protein by the MetAP. Thus, instead of possible involvement of other proteases, we suggest that the E. coli MetAP may possess other aminopeptidase activities for the terminal Ala, Leu, Gln, Phe, and Trp (Ben-Bassat et al. 1987). The removal of N-terminal bulky residues, such as Leu, Gln, Phe, and Trp, and the subsequent exposure of small residues, such as Ala, Ser, and Thr, may protect the processed proteins from degradation. As predicted from the N-end rule of protein stability, proteins possessing a bulky N-terminal residue, known as destabilizing residues, have short half-lives and those possessing small terminal residues, known as stabilizing residues, have long half-lives (Tobias et al. 1991; Varshavsky 1996).
In addition to Met removal, modification to the N-terminal residue is often crucial for the function of a protein. For example, the acetylation of the N-terminal Asp/Glu residue of actin strengthens its interaction with myosin (Abe et al. 2000). Also, the N-myristoylation of the N-terminal Gly of SOS3 protein is essential for conferring salt tolerance in plants (Ishitani et al. 2000). The conversion of the N-terminal Gln to Pyr is found in all known cytotoxic frog ribonucleases, some mammalian ribonucleases, immunoglobulins, and several hormones (thyrotropin releasing hormone, luteinizing hormone releasing hormone, gonadotropin-releasing hormone, corticotropin-releasing hormone, and gastrin [Busby Jr. et al. 1987; Fischer and Spiess 1987; Boix et al. 1996; Liao et al. 2003]).
Three hydrogen bonds contributed by N-terminal Pyr of frog ribonuclease are essential for its structural integrity, catalytic activity, and cytotoxicity (Huang et al. 1998; Leu et al. 2003; Liao et al. 2003). In this study, a large amount of demethioninylated proteins (10~50 mg from 1 L of culture) was prepared in vivo by engineered MetAP, and the terminal Gln was subsequently converted to Pyr in vitro under mild conditions instead of using chemical and enzymatic treatments (Shapiro et al. 1988; Notomista et al. 1999; Liao et al. 2003). For prevention of subsequent cleavage of the penultimate residue, the antepenultimate residue of any protein of interest may be replaced by bulky or acidic residues if they do not alter the properties of the protein, such as RCL1-N2D. Thus, the engineered MetAP and the expression system developed here in E. coli BL21(DE3) are able to produce large quantities of soluble or insoluble authentic protein.
The wild-type MetAP exhibited high catalytic activities for MAD-initiated proteins in vivo and for MAD-initiated oligopeptides in vitro. However, the engineered MetAP-*TG demonstrated high catalytic activities only for MQD-initiated proteins in vivo, but not for MQD-initiated oligopeptides in vitro. Although yeast MetAP I has been found to be associated with ribosomal proteins and mammalian MetAP II is associated with eukaryotic translation initiation factor (eIF-2; Wu et al. 1993; Vetro and Chang 2002), the possible association of cellular protein(s) with E. coli MetAP is not known. The MetAP-*TG retains high catalytic activity for MAD-initiated oligopeptides in vitro, but it does not exhibit significant catalytic activity for MQD-initiated oligopeptides. This is probably due to the relatively rigid structure of the purified enzyme and the limited space for the binding of bulky penultimate residues in vitro. In contrast, it may exhibit a higher flexibility in binding bulky penultimate residues if it is assisted by cellular proteins in vivo. Therefore, it is possible that the removal of terminal Met from bulky penultimate residues by E. coli MetAP may be assisted by other cellular protein(s) in vivo during the elongation phase of protein synthesis.
The amino acid residues responsible for catalytic activity and substrate specificity of MetAP are conserved in most living organisms (Tahirov et al. 1998), for example, the Met 206 and Gln 233 of E. coli MetAP and the Met 329 and Gln 356 of yeast MetAP I (Ben-Bassat et al. 1987; Hirel et al. 1989; Hwang et al. 1999). Thus, mutations of the corresponding residues of human MetAP will likely destroy its function. Furthermore, mutations of the penultimate or antepenultimate residues of any desired target protein may also alter their stability and function. Therefore, both of these features may provide important clues in the study of human genes and associated diseases and may help investigators explore the possibility of gene therapy, such as for hemoglobin Long Island with reduced oxygen affinity, which is caused by the His-Pro substitution at the third residue of -globin and the failure to remove the N-terminal Met (Prchal et al. 1986).
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Materials and methods |
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, PDB code 3MAT
[PDB]
) and amino acid sequence alignment of MetAPs (Ben-Bassat et al. 1987; Lowther et al. 1999; Walker and Bradshaw 1999). For the expression of secretory ribonucleases, the ribonuclease gene was cloned and expressed as described previously (Huang et al. 1998).
Expression and purification of MetAP and target proteins
The MetAP was expressed alone or coexpressed with a target protein, that is, onconase and RC-RNaseL1, in E. coli BL21 (DE3) in the presence of 0.5 mM isopropyl--D-thiogalactopyranoside at 37°C. After sonication, the MetAP in soluble fraction was dialyzed against buffer A (20 mM HEPES at pH 8.0, 50 mM KCl) and purified to homogeneity by phosphocellulose (Whatman P-11) and DEAE-cellulose (Whatman DE52) column chromatographies with a yield of 50 mg from 1 L of culture.
Most of the coexpressed target protein, for example, onconase or RC-RNase L1, existed in insoluble cell lysate. These proteins were denatured, renatured, and purified as described previously (Liao et al. 2003). The purified ribonucleases were stored in 20 mM sodium phosphate (pH 7.0) for Pyr formation at the N terminus. Alternatively, the ribonuclease secreted into culture medium through the pelB system was concentrated and purified to electrophoretical homogeneity with a yield of 1~5 mg per 1 L of culture (Huang et al. 1998).
Analysis of N termini of recombinant proteins
Proteins in the cell pellet or in the soluble fraction were separated by 13.3% SDS-PAGE and contact-transferred to ProBlott membrane (Applied Biosystems) in transfer buffer (10 mM Tris-HCl at pH 7.5, 50 mM NaCl, 2 mM EDTA, 0.5 mM 2-mercaptoethanol) overnight. The N-terminal five residues of the transferred protein were determined by Edman degradation. The percentage of Met removal was calculated by dividing the amount of protein initiated with the penultimate residue by the amount of total proteins (i.e., proteins initiated with the penultimate residue and unprocessed protein initiated with Met) after the first degradation cycle. The same calculation was made from five consecutive degradation cycles, for example, MQNWL for onconase. The efficiency of Met removal is reported as the mean values of at least three cycles. The mass spectrum analysis was carried out as described previously (Liao et al. 2003).
In vitro MetAP activity assay
The oligopeptide substrates used in this experiment were synthesized on the 432A Synergy peptide synthesizer (Applied Biosystems). The enzymatic activity of purified MetAP was assayed as described by Ben-Bassat et al. (1987). Briefly, the diluted MetAP enzyme in 10 µL was added to 90 µL of the substrate solution containing 4 mM oligopeptide, 0.1 M potassium phosphate buffer (pH 7.5), and 0.2 mM CoCl2, incubated for 10 min at 37°C and stopped in boiling water for 2 min. After addition of 0.9 mL of color development mixture containing 0.2 mg of L-amino acid oxidase, 0.03 mg of horseradish peroxidase, and 0.2 mg of o-dianisidine in 0.1 M Tris-HCl (pH 7.4), the tubes were incubated for 10 min at 37°C and the optical density at 440 nm was recorded. One unit of activity is defined as 1 µmole of amino acids produced per min under the assay conditions and the pure Met was used as standard. The N-terminal residues of MetAP-treated oligopeptide were further verified by Edman degradation.
Ribonuclease activity assay
Ribonuclease activity was analyzed by zymogram assay on RNA-casting PAGE (Liao and Wang 1994). In addition, the ribonuclease activity of each purified ribonuclease was determined by the release of acid-soluble nucleotides from bakers’ yeast total RNA after ribonuclease digestion. One unit of enzyme activity was defined as the amount of enzyme producing one A260 acid-soluble material for 15 min at 37°C (Huang et al. 1998).
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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 A. N. Suhasini and R. Sirdeshmukh Transfer RNA Cleavages by Onconase Reveal Unusual Cleavage Sites J. Biol. Chem., May 5, 2006; 281(18): 12201 - 12209. [Abstract] [Full Text] [PDF]
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 N. Shibuya, T. Nishiyama, and N. Nakashima Cell-Free Synthesis of Polypeptides Lacking an Amino-Terminal Methionine by Using a Dicistroviral Intergenic Internal Ribosome Entry Site J. Biochem., November 1, 2004; 136(5): 601 - 606. [Abstract] [Full Text] [PDF]
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