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Lack of the Consensus Sequence Necessary for Tryptophan Prenylation in the Comx Pheromone Precursor

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Lack of the Consensus Sequence Necessary for Tryptophan Prenylation in the Comx Pheromone Precursor Biosci. Biotechnol. Biochem., 76 (8), 1492–1496, 2012 Lack of the Consensus Sequence Necessary for Tryptophan 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 protein known to contain prenylated trypto- Apart from ComX, however, no other proteins 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 cysteine 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 Rab 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, farnesyltransferase; 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 mgmLÀ1; and Gln, 0.4 mg mLÀ1) and ampicillin (400 mgmLÀ1). 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- TrpÃ(Ger) represents the geranyl-modified tryptophan residue (bold coltetraacetic acid (EGTA) (buffer A). The slurry was ultra-centrifuged lines). B, Amino acid 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 mLÀ1) 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 À80 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 Â 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 minÀ1. 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.
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