From the
Previous work from this laboratory has shown that a derivative
of [
A number of proteins such as Ras protein, nuclear lamins, the
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
[
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
Pursuing metabolic labeling of H. cutirubrum cells with [
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
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.
-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.
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.
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.
, 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
(C
H
O
S,
C
H
OCH
CH(OC
H
)CH
SCH
)
and peaks at m/z 667 (M-CH
), 620
(M-CH
SCH
), 415
(M-C
H
), 384
(M-C
H
OH), 337
(M-C
H
OH-SCH
), and 278
(C
H
) (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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.