Biosci. Biotechnol. Biochem., 76 (8), 1492–1496, 2012

Lack of the Consensus Sequence Necessary for Prenylation in the ComX Pheromone Precursor

y Fumitada TSUJI,1; Ayako ISHIHARA,1 Aya NAKAGAWA,1 Masahiro OKADA,2 Shigeyuki KITAMURA,1 Kyoko KANAMARU,1 Yuichi MASUDA,3 Kazuma MURAKAMI,3 Kazuhiro IRIE,3 and Youji SAKAGAMI1

1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-8601, Japan 2Graduate School of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi 487-8501, Japan 3Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received March 19, 2012; Accepted May 7, 2012; Online Publication, August 7, 2012 [doi:10.1271/bbb.120206]

ComX, an oligopeptide pheromone that stimulates the pheromones from other strains also contain either natural genetic competence controlled by quorum geranyl- or farnesyl-modified tryptophan residues. sensing in Bacillus subtilis and related bacilli, contains Prenyl post-translational modification is essential for a prenyl-modified tryptophan residue. Since ComX is the biological activity of the ComX pheromone.5–8) the only known to contain prenylated trypto- Apart from ComX, however, no other contain- phan, the universality of this unique posttranslational ing prenylated tryptophan residues have so far been modification has yet to be determined. Recently, we identified. developed a cell-free assay system in which the trypto- Prenylation (farnesylation and geranylgeranylation) phan residue in the ComXRO-E-2 pheromone precursor of proteins on residues is a well-known post- derived from B. subtilis strain RO-E-2 can be gerany- translational modification. Prenyl modification of cys- lated by the ComQRO-E-2 enzyme. We report here our teine residues was first discovered in the oligopeptide attempt to identify the consensus sequence surrounding pheromones that induce conjugation tube formation in the geranylated tryptophan residue by using the cell- Basidiomycota, and is now recognized as a universal 9–11) free system with various ComXRO-E-2 pheromone pre- post-translational modification in eukaryotes. The cursor analogs. We found that [47–58]ComXRO-E-2, consensus sequence surrounding prenylated cysteine corresponding to the C-terminal 12-residue peptide of residues has also been determined,12) and consists of a the pheromone precursor, contained a short sequence CaaX motif, where ‘‘a’’ designates an aliphatic amino essential for geranylation. We also found that the length acid and ‘‘X’’ designates residues that are specific to of the sequence between the tryptophan residue and the particular prenyltransferases (FTase and GGTase-I). C-terminus was important for geranylation, and that While the consensus sequence has been found in some [47–58]ComXRO-E-2 pheromone precursor amino most prenylated proteins, some proteins possess no acids were involved in the geranylation reaction. How- consensus sequence such as which is prenylated by ever, we could not identify a consensus sequence RabGGTase.13) The prenylation of cysteine residues is surrounding the geranylated tryptophan. Our evidence essential for the biological functions and localization of suggests that, like Rab which lacks a consensus sequence a number of proteins; for example, the Ras oncoprotein yet is geranylgeranyl-modified on a cysteine residue, requires farnesylation in order to transform cells.14) As a the ComX pheromone and its precursor also lack a result, FTase inhibitors are attractive targets for cancer consensus sequence. therapy.15) Many prenylated proteins play important roles in many cells, so that considerable research using a Key words: ComX pheromone; post-translational mod- variety of methods has been focused on a comprehensive ification; prenylation; prenyltransferase; identification of the ‘‘prenylome.’’16,17) Bacillus subtilis Since prenylation of cysteine residues is a universal phenomenon, there is a strong possibility that the Bacillus subtilis and related bacilli develop compe- prenylation of tryptophan residues would also be a tence to DNA transformation for survival in response universal post-translational modification. Determining to environmental changes. The existence of a signal that the prenylation of tryptophan residues is indeed a substance that stimulates natural genetic competence universal phenomenon should lead to the discovery of was proposed in 1967, and the ComX pheromone was new prenylated proteins. isolated in 1994.1,2) The ComX pheromone is an A number of methods are available for identifying oligopeptide containing two types of prenyl modification novel proteins containing prenylated tryptophan resi- on tryptophan residues: geranyl modification (as found dues, such as screening with antibodies specific to 3) in ComXRO-E-2; Fig. 1A) and farnesyl modification (as prenylated tryptophan. Although our group has attempted 4) found in ComXRO-C-2). It is believed that ComX to produce an antibody specific to geranylated trypto-

y To whom correspondence should be addressed. Fax: +81-52-789-4118; E-mail: fumitada [email protected] Abbreviations: Ger, geranyl; FTase, ; GGTase-I, geranylgeranyltransferase type I; RabGGTase, Rab geranylgeranyltransferase; TAPS, N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; REP, Rab escort protein; CBR, C-terminus binding region; CIM, CBR interacting motif; SCRs, sequence-conserved regions Deduced Consensus Sequence of Geranylated Tryptophan 1493 A containing pET-15b-derived plasmids was grown in an M9 minimal medium supplemented with a mixture of L-amino acids (Leu, Phe, His, Met and Ser, 40 mgmL1; and Gln, 0.4 mg mL1) and ampicillin (400 mgmL1). DNA manipulation, cloning and standard molecular biological procedures were performed by the standard protocols.

Expression and preparation of ComQRO-E-2. Plasmid construction, expression, and preparation of ComQRO-E-2 were carried out as previously described.20) E. coli BL21(DE3) producer cells containing pET-15b-derived plasmids were grown in 100 mL of an M9 minimal medium supplemented with a mixture of L-amino acids and ampicillin B (see above), until OD620 had reached between 0.4 and 0.6. Over- expression of ComQRO-E-2 was induced by adding isopropyl -D- thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 4 h of induction, the precipitate was collected by centrifugation for Fig. 1. Chemical Structure of the ComXRO-E-2 Pheromone and 10 min at 10,000 rpm at 4 C. The precipitate was homogenized by Sequence of Its Precursor. sonication (60 W for 1 min, 5 times) at 4 C in 1.5 mL of a 25 mM Tris– A, Chemical structure of the ComXRO-E-2 pheromone. Bold HCl buffer (pH 7.4) containing 0.1 mM MgCl2, 0.1 mM ethylenegly- Trp(Ger) represents the geranyl-modified tryptophan residue (bold coltetraacetic acid (EGTA) (buffer A). The slurry was ultra-centrifuged lines). B, sequence of the ComXRO-E-2 pheromone at 38,000 rpm for 2 h at 4 C. The resulting precipitate was dissolved in precursor. Bold W and the underlined section respectively represent 400 mL of buffer A containing dodecyl -D-maltoside (2 mg mL1) the geranyl-modified tryptophan residue and mature ComXRO-E-2 (buffer B). The prepared membrane fraction of the crude enzyme pheromone. (ComQRO-E-2) was frozen and stocked at 80 C until needed.

In vitro geranylation and detection of the geranylated peptides. phan, we have been unable to obtain an antibody of In vitro geranylation and detection of the geranylated peptides were sufficient titer (data not shown). Another method for performed as described in a previous report.20) Triplicate samples detecting proteins prenylated on tryptophan is to define containing 50 mM ComXRO-E-2 precursor peptide, 200 mM geranyl the consensus sequence recognized by the modifying pyrophosphate ammonium salt (Sigma-Aldrich, St. Louis, MO, USA), and crude ComQRO-E-2 (7.5 mL of the broth eq.) were incubated enzyme, ComQ, and then to identify the prenylated proteins by in silico database searching on the basis of for 2 h at 37 Cina50mL final volume of a 50 mM TAPS-NaOH buffer (pH 8.5) containing 5 mM MgCl . The enzyme reaction was stopped by the consensus sequence. However, the extreme poly- 2 chilling the samples in an ice bath and adding 200 mLofCH3CN to the morphism (only the modified tryptophan residue is reaction mixture. The mixture was centrifuged at 15,000 rpm for 5 min conserved) in the mature ComX pheromone18,19) has at 4 C, and the resulting supernatant was mixed with an internal 7) made difficult to deduce the consensus sequence. Since standard (250 mM Ala-ComXRO-E-2) and analyzed by LC-MS the ComX precursor sequence contains several con- (HCTplus, Bruker Daltonics, Billerica, MA, USA), using a Develosil served amino acids, the consensus sequence is likely C30-UG-5 column (0:3 150 mm, Nomura Chemical, Seto, Japan) to exist in the precursor sequence rather than in the and eluting with a linear gradient of 15–80% CH3CN in H2O containing 0.1% formic acid. The chromatographic flow rate was set at mature sequence, although this possibility has not been 5.0 mL min1. The geranylated peptides were confirmed by comparing confirmed. the chromatographic retention time and MS and MS/MS data with We have recently developed a cell-free system by those of synthetic geranylated peptides. The geranylated peptides were which the tryptophan residue in the [1–58]ComXRO-E-2 quantified by using a standard curve for the chemically synthesized pheromone precursor (Fig. 1B) is modified with a [52–58]ComXRO-E-2 pheromone. This standard curve was drawn by 20) using 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM,1mM and geranyl group by a crude ComQRO-E-2 enzyme. This 2 mM of the chemically synthesized [52–58]ComX pheromone system may be useful for characterizing the universality RO-E-2 with an internal standard (250 mM Ala-ComXRO-E-2). The amount of the of tryptophan prenylation. We deduced in the present geranylated peptide was calculated from integration of the peak area of study the sequence necessary for geranylation in ComX the mass chromatogram, using the standard curve just mentioned. The by using the in vitro cell-free system with truncated and yield of the in vitro enzyme reaction is expressed as a percentage of the substituted ComXRO-E-2 pheromone precursors. We geranylated product to the non-geranylated starting material. found that the [47–58]ComXRO-E-2 pheromone precur- sor, a peptide consisting of 12 residues from the Results and Discussion C-terminus, contained a short sequence essential for geranylation. We also found that the Trp residue must lie Identification of the short sequence essential for between the 2–4 residues from the C-terminus and geranylation identified the other resides essential for prenylation. We first determined the short sequence essential for geranylation by using an in vitro geranylation system Materials and Methods with various N-terminal-truncated ComXRO-E-2 phero- mone precursors (Supplemental Table S1; see the

Peptide synthesis. All ComXRO-E-2 precursor analogs were synthe- Biosci. Biotechnol. Biochem. Web site). Geranylated sized by either a Pioneer (Applied Biosystems, Foster City, CA, USA) peptides were obtained in a high yield with various or 433A peptide synthesizer (Applied Biosystems) by using Fmoc truncated ComXRO-E-2 pheromone precursors spanning 8,20,21) chemistry as previously described. N-terminal residues 1–58 through 47–58 (Fig. 2). In contrast, the yield of geranylated peptides was very low Growth conditions and general methods. Escherichia coli DH5 with various truncated ComXRO-E-2 pheromone precur- (Takara Bio, Otsu, Japan) was used for cloning the comQRO-E-2 gene into the pET-15b vector (Novagen), and the transformant was selected sors spanning N-terminal residues 48–58. These results on Luria-Bertani (LB) agar supplemented with ampicillin indicated that the short sequence essential for geranyl (50 mgmL1). The E. coli BL21(DE3) (Takara Bio) producer cell modification was located at the C-terminus of the 1494 F. TSUJI et al. A

B Fig. 2. Determination of the Short Sequence Necessary for Gerany- lation. The yield is shown of the geranylated peptides produced by the in vitro cell-free enzyme system using N-terminal truncation of the ComXRO-E-2 pheromone precursor.

ComXRO-E-2 pheromone precursor between residues 47 and 58.

In vitro geranylation using [47–58]ComXRO-E-2 pher- omone precursor analogs Fig. 3. Verification of the Importance of the C-Terminal Sequence and Identification of the Amino Acids Necessary for Geranylation. We next performed in vitro geranylation experiments A, Yield of geranylated peptides produced by the in vitro cell-free by using various C-terminal-modified and - enzyme system using C-terminal-modified analogs. B, Yield of substituted [47–58]ComXRO-E-2 analogs to locate the geranylated peptides produced by the in vitro cell-free enzyme sequence necessary for geranylation (Supplemental system using alanine-substituted analogs. Asterisks represent the geranylated peptides produced in a yield of less than one-third the Table S2; see the Biosci. Biotechnol. Biochem. Web yield of the positive control (the [47–58]ComXRO-E-2 pheromone site). The C-terminal-modified analogs consisted of four precursor). Underlined residues represent the amino acids involved peptides with either truncated C-termini or an extension in geranylation of the [47–58]ComXRO-E-2 pheromone precursor. of one or two alanine residues at the C-terminus. We performed in vitro geranylation by using these analogs located in the vicinity of the C-terminus after processing. and calculated the yield of geranylated peptides pro- The mature sequence of such ComX pheromones as duced in order to verify the importance of the C-terminal ComXNAF4 must therefore be identified in order to sequence. Since the main product was a geranylated confirm the importance of the length of the sequence peptide derived from cleavage between cysteine 51 and between the tryptophan residue and the C-terminus. 52, we calculated the yield of the geranylated A total of 11 alanine-substituted analogs was used, in peptide corresponding to the C-terminal sequence of which an alanine residue was substituted at every each analog (e.g., in the case of the [47–56]ComXRO-E-2 position except for that of the tryptophan residue. We analogs, [52–56]ComXRO-E-2 was the product). The used these analogs to perform in vitro geranylation and analogs consisting of either a deletion or extension of identify the amino acid(s) involved in geranylation. We one residue consequently led to a decline in the yield of calculated the total yield for all geranylated peptides geranylated peptides by almost half, and the yield of detected by an LC-MS analysis, because of concerns geranylated peptides produced by using analogs consist- regarding alanine substitution not only affecting ger- ing of either a deletion or extension of two residues was anylation but also processing. A significant decrease in almost zero (Fig. 3A). This result indicated that the yield the yield of particular analogs indicated that 47, of geranylated peptides depended upon the length of the cysteine 51, isoleucine 54, phenylalanine 55, and glutamic chain between the geranylated tryptophan residue and the acid 57 were important for geranylation (Fig. 3B). C-terminus, and that the tryptophan residue must be located at least in the second, third, or fourth position Deducing the consensus sequence surrounding the from the C-terminus in order for geranylation to occur. geranyl-modified tryptophan residue Our result is in agreement with a previous report We compared the sequences of the first 12 C-terminal indicating that the tryptophan residue was located in residues of ComX pheromone precursors from several the third or fourth position from the C-terminus in all of strains (Fig. 4). Of the residues that alanine substitution the ComX pheromones for which mature sequences have experiments indicated to be important for geranylation, been reported.22) Our results also suggested that ComQ only the leucine residue was conserved. For example, could distinguish the chain length between the modified the phenylalanine residue, which affected the efficiency tryptophan residue and the C-terminus in ComX pher- of geranylation more than any other residue, was not omone precursors. However, in such ComX pheromone conserved at all. Cys was also important for modifica- precursors as ComXNAF4 from Bacillus subtilis (natto) tion, but it is unclear at this time whether the NAF4, the tryptophan residue is not located in the third nucleophilic amino acid was one of the consensus or fourth position from the C-terminus.23) The mature residues, together with Ser and Thr in other ComX sequence of such ComX pheromones has not been pheromones, or played a specific role in geranylation of reported, and the modified tryptophan residue can be the RO-E-2 strain. It was therefore not possible to Deduced Consensus Sequence of Geranylated Tryptophan 1495

Fig. 4. Alignment of the 12-Residue C-Terminal Sequences of Various ComX Pheromone Precursors. Fig. 6. Verification of the Importance of the Length of the Sequence Bold W represents the geranyl- or farnesyl-modified tryptophan between the Conserved Leucine Residue and the Target Tryptophan residue. Asterisks denote conserved residues. The sequences were Residue. aligned by using ClustalW (http://www.uniprot.org/). Yield of geranylated peptides produced by the in vitro cell-free enzyme system using variable-length-sequence [47–58]ComXRO-E-2 pheromone precursor analogs. Peptides with a geranyl modification A 7-, 6-, and 5-residues from the C-terminus were detected.

modifying enzyme, RabGGTase, it did contain two binding sites recognized by the adapter protein, REP, B which mediates recognition by RabGGTase.13) The first REP binding site was in the conserved Rab GTPase domain in the vicinity of the N-terminus, while the second binding site was within a conserved sequence of 10–15 amino acids upstream from the cysteine residue to be prenylated in the CBR interacting motif (CIM). The length of the sequence between CIM and the target cysteine residue is crucial for prenylation.24) The ComX pheromone precursor had some similarities to Rab. First, the ComX pheromone precursor also contained a well- conserved domain in the vicinity of the N-terminus and extreme polymorphism in the C-terminal sequence. Fig. 5. In Vitro Geranylation Experiment Using the [42– Second, the enzymatic activity of both ComQ and 53]ComXRO-H-1 and [43–54]ComXRO-B-2 Pheromone Precursors. RabGGTase required the magnesium ion rather than A, Sequences of the [42–53]ComXRO-H-1 and [43–54]ComXRO-B-2 14,20) pheromone precursors, both of which consisted of the first 12 zinc ion. Third, the well-conserved leucine residue, residues from the C-terminus. B, Yield of geranylated peptides which was located nine residues upstream from the produced by the in vitro cell-free enzyme system using the [42– tryptophan residue to be prenylated, and CIM, which 53]ComXRO-H-1 and [43–54]ComXRO-B-2 pheromone precursors. was located 10–15 residues upstream from the cysteine residue to be prenylated, were both crucial for preny- deduce the consensus sequence surrounding the geranyl- lation. In addition, the length of the sequence between modified tryptophan residue by using this approach. the conserved leucine residue and the target tryptophan We then hypothesized that if a consensus sequence residue, and the length of the sequence between CIM were to exist, ComQRO-E-2 should geranylate other and the target cysteine residue were both crucial for ComX pheromone precursors as it geranylated prenylation24) (Fig. 6, Supplemental Table S3; see the ComXRO-E-2. We therefore performed an in vitro Biosci. Biotechnol. Biochem. Web site). These results geranylation experiment using the [42–53]ComXRO-H-1 suggest that the conserved leucine residue (including its and [43–54]ComXRO-B-2 pheromone precursors, both surrounding amino acid, perhaps) was involved in of which consisted of the first 12 residues from the binding to an adapter protein like CIM. Taken together, C-terminus (Fig. 5A). Although the reaction mixtures these data suggest that, like Rab, the ComX pheromone were analyzed by using LC-MS, no geranylated peptides precursor contained no consensus sequence. However, could be detected (Fig. 5B), suggesting that ComQRO-E-2 no adapter protein analogous to REP has so far been recognized a specific C-terminus of the ComXRO-E-2 identified for ComX. It has been shown that REP sequence, and that the ComXRO-H-1 and ComXRO-B-2 contained several sequence-conserved regions pheromone precursors possessed the conserved C- (SCRs);25) however, no SCR-containing proteins have terminus amino acid residue, Leu, as well as the been found in Bacillus subtilis by database searching. If ComXRO-E-2 pheromone precursor. These results implied a ComX pheromone precursor adapter protein were to that there was no consensus sequence surrounding the exist, it would therefore be a novel protein with a geranyl-modified tryptophan residue in the ComX different structure from that of REP. pheromone precursor. Among proteins that are prenylated on the cysteine Conclusions residues, some such as Rab have been found to lack the consensus sequence necessary for prenylation. Although We used an in vitro cell-free enzyme reaction system Rab contained no consensus sequence recognized by the with various truncated ComXRO-E-2 pheromone precur- 1496 F. TSUJI et al. sors to find that the [47–58]ComXRO-E-2 pheromone 4) Okada M, Yamaguchi H, Sato I, Tsuji F, Dubnau D, and precursor, a peptide consisting of 12 residues from the Sakagami Y, Biosci. Biotechnol. Biochem., 72, 914–918 (2008). C-terminus of ComX , contained a short sequence 5) Okada M, Sato I, Cho SJ, Suzuki Y, Ojika M, Dubnau D, and RO-E-2 Sakagami Y, Biosci. Biotechnol. Biochem., 68, 2374–2387 essential for geranylation. Examining various [47– (2004). 58]ComXRO-E-2 pheromone precursor analogs by using 6) Okada M, Sato I, Cho SJ, Dubnau D, and Sakagami Y, the in vitro cell-free system indicated that the length of Tetrahedron, 62, 8907–8918 (2006). the sequence between the tryptophan residue and the 7) Okada M, Yamaguchi H, Sato I, Cho SJ, Dubnau D, and C-terminus was important for geranylation, and that Sakagami Y, Bioorg. Med. Chem. Lett., 17, 1705–1707 (2007). some of these amino acids were involved in the 8) Tsuji F, Kobayashi K, Okada M, Yamaguchi H, Ojika M, and geranylation reaction. However, comparing the ComX Sakagami Y, Bioorg. Med. Chem. Lett., 21, 4041–4044 (2011). 9) Kamiya Y, Sakurai A, Tamura S, and Takahashi N, Biochem. pheromone precursors from other strains based upon Biophys. Res. 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