©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Type of Protein Modification by Isoprenoid-derived Materials
DIPHYTANYLGLYCERYLATED PROTEINS IN HALOBACTERIA(*)

Hiroshi Sagami (§) , Akihiro Kikuchi , Kyozo Ogura (§)

From the (1)Institute for Chemical Reaction Science, Tohoku University, 2-1-1, Katahira, Sendai 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Previous work from this laboratory has shown that a derivative of [H]mevalonic acid is incorporated into a number of specific proteins in Halobacterium halobium and Halobacterium cutirubrum and that the major radioactive material released by treatment with methyl iodide was neither farnesyl nor geranylgeranyl compound, which have been generally accepted to be prenyl groups of a number of prenylated proteins found in eukaryotic cells, but an unknown compound (Sagami, H., Kikuchi, A., and Ogura, K.(1994) Biochem. Biophys. Res. Commun. 203, 972 -978). In the current study, the unknown compound was prepared in a large amount from H. halobium cells and analyzed by reverse and normal phase high performance liquid chromatographies followed by mass spectrometry. The mass spectrum of this compound exhibited a parent ion peak (M) at m/z 682, suggesting that it is a 1-methylthio-2,3-di-O-(3`,7`,11`,15`-tetramethylhexadecyl)glycerol (diphytanylglyceryl methylthioether). Diphytanylglyceryl methyl thioether was chemically synthesized, and its mass fragmentation pattern was completely coincident with that of the mevalonic acid-derived material from H. halobium. These results indicate that Halobacteria contains specific proteins with a novel type of modification of a cysteine residue of the proteins with a diphytanylglyceryl group in thioether linkage.


INTRODUCTION

A number of proteins such as Ras protein, nuclear lamins, the -subunits of large molecular G-proteins, various Ras-like low molecular weight G-proteins, and heat shock proteins in eukaryotic cells have been found to be modified with isoprenoids such as farnesyl and geranylgeranyl groups(1, 2, 3, 4, 5, 6, 7) . The isoprenoid is linked to a carboxyl-terminal cysteine of the proteins through thioether linkage. The prenylation is indispensable for the proteins to play the role in signal transduction, cell cycle progression, transformation, and protein transportation. Epstein et al.(8) have revealed that the amount of prenylated proteins in prokaryotic cells is much lower than that in eukaryotic cells. In particular, no prenylated proteins have been detected in Escherichia coli. It is very important to see whether prokaryotic cells contain such prenylated proteins responsible for the fundamental cellular functions as found in eukaryotic cells or any other lipid-linked proteins.

To investigate whether or not prokaryotic cells have proteins modified by mevalonic acid-derived materials, we previously performed metabolic labeling of extremely halophilic archaebacteria, Halobacterium halobium and Halobacterium cutirubrum, with [H]mevalonic acid and found that mevalonic acid-derived materials were actually incorporated into a number of proteins on SDS-polyacrylamide electrophoresis followed by fluorography(9) . We also pointed out that the material released by treatment of delipidated proteins with methyl iodide is neither farnesyl nor geranylgeranyl but an unknown compound that migrated on a reverse phase (C) thin layer plate with a similar mobility to that of a prenol with a carbon chain length of C. In this study, the released material was further purified by reverse and normal phase high performance liquid chromatographies (HPLC)()and analyzed by mass spectrometry (MS). As a result, the released material was determined to be a diphytanylglyceryl methyl thioether.


EXPERIMENTAL PROCEDURES

Materials

R-[5-H]Mevalonic acid (34.3 Ci/mmol) was purchased from DuPont NEN. Polyprenols (C-C) from Ginkgo biloba and E,E,E-geranylgeraniol (C) were generous gifts from Kuraray Corp. Diphytanylglycerol was prepared from H. halobium cells according to the method of Minnikin et al.(10) and confirmed by MS(11) . All other chemicals were from commercially available sources.

Release of Radioactive Materials from Delipidated Proteins

Cells of H. halobium were grown as described in a previous report(9) . Cells were labeled for 96 h with 0.15 µM [5-H]mevalonic acid, combined with unlabeled cells (wet weight, 25 g), washed, and extracted with acetone:water (4:1), chloroform:methanol (2:1), chloroform:methanol:water (10:10:3), and ethanol. The delipidated proteins were then treated with methyl iodide as described previously (9). The recovery of [H]mevalonic acid-derived materials was maximally 40%.

HPLC Purification of the Released Radioactive Materials

The radioactive materials released by the treatment of delipidated proteins with methyl iodide were purified by silica gel Sep-Pak chromatography (Waters) in a solvent system of methylene chloride. The radioactive materials were recovered 70% in run-through fractions. The run-through fractions were combined and concentrated to dryness with nitrogen and purified by reverse phase C HPLC (YMC, 250 4.6 mm) in acetone:water (30:1). The radioactive materials were further purified by silica gel HPLC (YMC, 250 4.6 mm) in a solvent system of hexane:methylene chloride (5:1) before MS analysis.

MS and NMR Analysis

MS spectra were recorded on a JEOL JMS-DX300 mass spectrometer. Samples were introduced on a probe at 200 °C directly into the ion source. The potential of the ionizing beam was 70 eV. NMR spectra were measured with a Bruker NMR spectrometer (H NMR, 300 MHz). Samples were dissolved in CDCl. Chemical shifts were given in units relative to tetramethylsilane used as an internal standard. Splitting patterns were designated as s (singlet) and m (multiplet).

Synthesis of Diphytanylglyceryl Methyl Thioether

Diphytanylglyceryl methyl thioether was synthesized according to a modified method of Kates et al.(12) from methyl thioglycerol and phytanyl bromide. Commercially available 1-thioglycerol (1 ml) and potassium hydroxide (0.76 g) were mixed with methyl iodide (0.72 ml) in ethanol, and the mixture was stirred at 0 °C under nitrogen for 15 min. The solvent was evaporated, and the products were purified by silica gel (Merck Kieselgel 60) column chromatography in a solvent system of chloroform:methanol (20:1). The yield was 86%. Thus, obtained methyl thioglycerol was confirmed by 300 MHz H NMR. The methyl thioglycerol (265 mg) was mixed with phytanyl bromide (1.8 g) in dry benzene, and to the mixture was added powdered potassium hydroxide (1.3 g). The mixture was heated under reflux with magnetic stirring for 36 h, the water being removed by means of a phase-separating head. The cooled mixture was then diluted with diethyl ether and successively washed with water. The crude oil obtained after evaporation of the solvent was chromatographed on normal phase (Merck Kieselgel 60) and reverse phase (Merck LiChroprep RP-18) columns in a solvent system of hexane:methylene chloride (5:1) and acetone:water (40:1), respectively. The fractions containing diphytanylglyceryl methyl thioether were pooled, concentrated, and dried under vacuum. The yield was 26%. The dried sample was analyzed by NMR: H NMR (300 MHz, CDCl) 0.82-0.95 (30H, m, methyls), 1.00-1.43 (40H, m, methylenes), 1.45-1.69 (8H, m, methines), 2.16 (3H, s, S-methyl), 2.59-2.78 (2H, m, S-methylene), 3.45-3.63 (7H, m, O-methylenes and O-methine). Analytical TLC was carried out using reverse phase (Whatman LKC-18) and normal phase (Merck Kieselgel 60) plates. All the samples on thin layer plates were visualized with iodine vapor or by spraying 10% phosphomolybdate methanol solution and heating the plates.


RESULTS AND DISCUSSION

To elucidate the structure of the material released by treatment of delipidated proteins with methyl iodide, we prepared a large amount of delipidated proteins from H. halobium cells and treated them with methyl iodide as described under ``Experimental Procedures.'' The released materials actually migrated on a reverse phase thin layer plate as if they were polyprenols with a chain length longer than C, but they moved faster than C polyprenols on a silica gel thin layer plate, suggesting that they are non-polar compounds. Concerning the fragments released by methyl iodide treatment of proteins modified by lipids through thioether linkages, there should be two possibilities depending on the lipid structure. One is the case of a lipid with an allylic double bond, the fragments being a mixture of primary and tertiary alcohols(13) . The other is the case of a lipid without an allylic double bond. In this case, the fragments would be a mixture of a primary alcohol and a methyl thioether compound. The non-polar nature of the unknown material released raised the possibility that it might be a methyl thioether. The released mevalonic acid-derived materials were purified by silica gel Sep-Pak column chromatography followed by reverse phase C HPLC (Fig. 1). The radioactive materials were a mixture with minor components. Therefore, the fractions (fractions 31-34) corresponding to the major radioactivity peak were combined and further purified by silica gel HPLC and analyzed by MS. We assumed that the purified material is a diphytanylglyceryl methyl thioether compound on the basis of the mass spectrum, which exhibited a parent ion at m/e 682 (CHOS, CHOCHCH(OCH)CHSCH) and peaks at m/z 667 (M-CH), 620 (M-CHSCH), 415 (M-CH), 384 (M-CHOH), 337 (M-CHOH-SCH), and 278 (CH) (Fig. 2A). The fragmentation is well explained as shown in Fig. 3. To confirm the structure of this compound, we chemically synthesized 1-methylthio-2,3-di-O-(3`,7`,11`,15`-tetramethylhexadecyl)glycerol (diphytanylglyceryl methyl thioether) and analyzed it by HPLC, NMR, and MS. As a result, the synthetic specimen co-migrated with the unknown compound on the analytical HPLC (data not shown). In addition, the unknown compound was in good agreement with the synthetic specimen with respect to MS spectrum (Fig. 2B). These results indicate that a diphytanylglyceryl group is attached to a cysteine of protein in thioether linkage.


Figure 1: Reverse phase C HPLC of the run-through fractions of silica gel Sep-Pak chromatography of mevalonic acid-derived materials released by methyl iodide treatment of delipidated proteins. Fractions (5 and 0.5 ml) were collected for fractions 1-5 and fractions 6-100 at a flow rate of 1.0 ml/min, respectively. An aliquot (40 µl) was counted in scintillation fluid.




Figure 2: Mass spectrograms of the material corresponding to the major peak in Fig. 1 (A) and authentic diphytanylglyceryl methyl thioether (B). The samples were introduced on a probe at 200 °C directly into the ion source. The potential of the ion beam was 70 eV.




Figure 3: Structure of mevalonic acid-derived materials released from delipidated proteins by treatment with methyl iodide.



Judging from the structure of diphytanylglyceryl cysteinyl thioether in the lipid-bound form, the treatment with methyl iodide would give either diphytanylglycerol and diphytanylglyceryl methyl thioether, or both. Therefore, we again analyzed the radioactive materials released from delipidated proteins in details by use of authentic diphytanylglycerol(10, 11) . However, no detectable amount of released diphytanylglycerol was formed. Presumably, diphytanylglyceryl methyl thioether must be the only major product in the cleavage reaction of the thioether. This reaction can be rationalized as shown in Fig. 4.


Figure 4: Possible mechanism for methyl iodide treatment.



In this report, we have shown the evidence for the diphytanylglycerylation of proteins for the first time. It presents a new type of protein modification by isoprenoid-derived material. Covalent thioether modification of a cysteine residue with a diacylglyceryl group has been already identified for several proteins such as lipoproteins found in E. coli(14) . In the biosynthetic pathway of diacylglycerylated proteins(15) , the glyceryl moiety in phosphatidylglycerol is transferred to the sulfhydryl group of the cysteine in Leu-X-Y-Cys sequences (X or Y indicates neutral small amino acid) following signal peptide sequences of the amino-terminal region of the precursor proteins(15) . The glyceryl proteins are further modified with the acyl moieties in phospholipids. Thereafter, a prolipoprotein signal peptidase removes the signal peptide by cleavage of the diacylglyceryl precursor protein at the position between Y and diacylglyceryl-Cys, and then the free terminal NH group of the protein is modified with an acyl moiety in phospholipids. In this case, the modification by the diacylglyceryl group is located at the amino-terminal cysteine of proteins (Fig. 5B). Archaebacteria have unique membrane lipids(11) , which are linked to phytanyl groups through ether linkages. Although there is a similarity in the chemical structure between the diacylglyceryl group in eubacteria and the diphytanylglyceryl group in archaebacteria as shown in the present study, it remains unclear whether the diphytanylglycerylation occurs on the amino terminus. Mattar et al.(16) have recently characterized halocyanin, an archaeal blue copper protein in a haloalkaliphilic archaeron Natronobacterium phraonis. They observed a discrepancy between the molecular mass of the unmodified halocyanin polypeptide deduced on the basis of the cDNA sequence and that of the copper-free endogenous halocyanin determined by electrospray MS. To explain the discrepancy, they presumed a diphytanylglycerylation of the sulfhydryl group of the amino-terminal cysteine in addition to removal of a signal peptide and acetylation of the NH group of the same cysteine, although the structural elucidation has not been made. Taking all these into account, we assume that the diphytanylglycerylation occurs at the amino-terminal cysteine of proteins (Fig. 5A). In addition, it would be reasonable to propose the acetylation of the terminal amino group from mechanistic consideration of the cleavage reaction as discussed in Fig. 4. It also seems likely that archaebacteria incapable of synthesizing fatty acids (17) modify the amino terminus by acetylation instead of fatty acylation.


Figure 5: Two types of covalent modification of a cysteine residue with a glyceride thioether group. A, diphytanylglycerylated proteins presented in this report are characteristic of archaebacteria (H. halobium). It is expected that the diphytanylglycerylation is located at the amino-terminal cysteine of proteins. B, diacylglycerylated proteins such as lipoprotein in E. coli are mainly found in eubacteria. Diacylglycerylation is located at the amino-terminal cysteine of proteins.



It is known that two enzymatic steps are involved in the diacylglycerylation of cysteines of proteins in eubacteria(15) . They are glycerylation and acylation, in which the lipid donors are phosphatidylglycerol and phospholipids such as phosphatidylethanolamine, phosphatidylglycerol, or cardiolipin, respectively. By analogy, it is possible to assume the first-step transfer of the glyceryl moiety in phosphatidylglycerol onto proteins. However, it is unlikely that the second-step phytanylation of glyceryl proteins occurs as an exchange reaction of the phytanyl group between archaebacterial phospholipids and glyceryl proteins, because the ether-linked phytanyl group in phospholipids is not so reactive as the ester-linked fatty acyl group in phospholipids in eubacteria. For the diphytanylation there must be a different pathway from that for diacylglycerylation in eubacteria.

Pursuing metabolic labeling of H. cutirubrum cells with [H]mevalonic acid, Moldoveanu and Kates (18) have reported that the biosynthetic pathway for the major diphytanylglyceryl ether phospholipids involves their respective allylic ether-linked isoprenyl intermediates such as geranylgeranyl and phytyl intermediates, which undergo stepwise reduction to form the final saturated phytanyl ether phospholipids. Further, Poulter's group (19, 20) has recently reported in vitro formation of digeranylgeranylglyceryl phosphate from glyceryl phosphate and two molecules of geranylgeranyl diphosphate in two-step mechanisms. These results suggest that geranylgeranylation occurs on the glycerylcysteine residue of precursor proteins in archaebacteria and that the transferred digeranylgeranyl moiety is further reduced to the corresponding diphytanyl structure. The minor components shown in Fig. 1might be derivatives from intermediate proteins modified with lipids such as the phytanylphytylglyceryl group and diphytylglyceryl group.

In a previous report (9) we have shown that the endogenous amount of prenylated proteins such as geranylgeranyl and farnesyl proteins of H. halobium cells was too low to detect. However, we have also reported enzymatic formation of farnesyl Ras protein from Ras precursor protein and farnesyl diphosphate by 100,000 g supernatant of this bacterium(9) . Coupled together, it is concluded that H. halobium contains at least two kinds of isoprenoid-modified proteins besides retinal-linked proteins such as bacteriorhodopsin(21) , diphytanylglycerylated proteins, and prenylated proteins, with the former being the major one. It is conceivable that these lipid-modified proteins, irrespective of the location of their modification, share a common requirement of anchoring an otherwise hydrophilic polypeptide to the membrane via hydrophobic lipid moieties in a structurally diverse group of proteins in order to function at the membrane/aqueous interface. Further experiments are in progress to identify the specific diphytanylglycerylated proteins and elucidate the biosynthetic pathway of the new lipoproteins in Halobacteria.


FOOTNOTES

*
This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. for Chemical Reaction Science, Tohoku University 2-1-1, Katahira, Aobaku, Sendai 980, Japan.

The abbreviations used are: HPLC, high performance liquid chromatography(ies); MS, mass spectrometry.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.