Evidence for Covalent Attachment of Diphytanylglyceryl Phosphate
to the Cell-surface Glycoprotein of Halobacterium
halobium*
Akihiro
Kikuchi,
Hiroshi
Sagami
, and
Kyozo
Ogura
From the Institute for Chemical Reaction Science, Tohoku
University, 2-1-1, Katahira, Aobaku, Sendai 980-8577, Japan
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ABSTRACT |
In a previous study, we demonstrated the
occurrence of novel proteins modified with a diphytanylglyceryl group
in thioether linkage in Halobacterium halobium (Sagami, H.,
Kikuchi, A., and Ogura, K. (1995) J. Biol. Chem. 270, 14851-14854). In this study, we further investigated protein
isoprenoid modification in this halobacterium using several radioactive
tracers such as [3H]geranylgeranyl diphosphate. One of
the radioactive bands observed on SDS-polyacrylamide gel
electrophoresis corresponded to a periodic acid-Schiff stain-positive
protein (200 kDa). Radioactive and periodic acid-Schiff stain-positive
peptides (28 kDa) were obtained by trypsin digestion of the labeled
proteins. The radioactive materials released by acid treatment of the
peptides showed a similar mobility to dolichyl (C55)
phosphate on a normal-phase thin-layer plate. However, radioactive
hydrolysates obtained by acid phosphatase treatment co-migrated not
with dolichol (C55-65), but with diphytanylglycerol on
both reverse- and normal-phase thin-layer plates. The mass spectrum of
the hydrolysate was also coincident with that of diphytanylglycerol.
The partial amino acid sequences of the 28-kDa peptides were found in a
fragment (amino acids 731-816) obtainable by trypsin cleavage of the
known cell-surface glycoprotein of this halobacterium. These results indicate that the cell-surface glycoprotein (200 kDa) is modified with
diphytanylglyceryl phosphate.
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INTRODUCTION |
Several specific proteins including Ras protein in eukaryotic
cells have been found to be modified with isoprenoids such as farnesyl
and geranylgeranyl groups in thioether linkage with cysteine residues
of the proteins at the carboxyl termini. These isoprenylated proteins
play important roles in various biological functions such as signal
transduction, cell cycle progression, transformation, and protein
transportation (1-3). Occurrence of these isoprenylated proteins has
also been reported in archaebacteria such as Methanobacterium thermoautotrophicum, though their amount is small compared with that in eukaryotic cells. On the other hand, no isoprenylated proteins
have so far been found in eubacteria such as Escherichia coli (4). To learn more about the isoprenoid-modified proteins, we
labeled cells of an extremely halophilic archaebacterium,
Halobacterium halobium, with [3H]mevalonic
acid and analyzed the radioactive lipid moiety of the resulting labeled
proteins. Analysis of the lipid moiety revealed that a
diphytanylglyceryl group was associated with the proteins in thioether
linkage (5, 6).
To understand the biosynthesis of diphytanylglycerylated proteins, we
tried to label halobacterial cells with several radioactive tracers in
addition to mevalonic acid because mevalonic acid was, in fact,
effectively incorporated into the cells, but the amount of the
resulting labeled proteins was too small to be analyzed further. In the
course of these experiments, we became aware of the degradation of
isoprenoid-modified proteins during cell lysis by the action of
endogenous proteases. We improved the analytical method for protein
preparation. In this work, we report another novel isoprenoid
modification of proteins occurring in this archaebacterium. We also
describe evidence for covalent attachment of this isoprenoid to the
halobacterial cell-surface glycoprotein.
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EXPERIMENTAL PROCEDURES |
Materials--
(R)-[5-3H]Mevalonic
acid,
[1-3H]GOH,1
[1-3H]FOH, [1-3H]GGOH,
[1-3H]GGP, and [1-3H]GGPP (each 15-20
Ci/mmol) were purchased from American Radiolabeled Chemicals. Dolichol
(C55) and wheat germ acid phosphatase were purchased from
Sigma. Dolichyl (C55) phosphate was chemically synthesized
from dolichol (C55) according to the method of Danilov and
Chojnacki (7). Dolichols (C60-65) were purified by silica
gel column chromatography of dolichols (C60-90) obtained from Ginkgo biloba seeds, followed by reverse-phase
C18 column chromatography. Diphytanylglycerol was prepared
from H. halobium cells according to the method of Minnikin
et al. (8) and confirmed by mass spectrometry (9). Trypsin
(code no. 109819) and Pronase (code no. 165921) were purchased from
Roche Molecular Biochemicals. All other chemicals were from
commercially available sources.
Culture Conditions--
H. halobium (R1M1 strain) was
a gift from Dr. N. Tokunaga (Osaka University). Cells were grown at
38 °C in an illuminated gyratory shaker (120 rpm) in a growth medium
containing 250 g/liter NaCl, 2 g/liter KCl, 20 g/liter
MgSO4·7H2O, 0.2 g/liter
CaCl2·2H2O, 3 g/liter trisodium citrate, and
10 g/liter bacteriological peptone (Oxoid). The pH was adjusted to 7.4 before autoclaving.
Metabolic Labeling--
H. halobium cells (20 ml)
were labeled in the presence of 5 µCi/ml [3H]mevalonic
acid, [3H]GOH, [3H]FOH,
[3H]GGOH, [3H]GGP, or
[3H]GGPP for 96 h at 38 °C. The cells were
collected by centrifugation at 10,000 × g for 10 min
at 4 °C and washed with a basal salt solution (growth medium without
peptone), followed by centrifugation for 10 min. The washed cells were
lysed in 5 ml of 10 mM Tris-HCl buffer (pH 7.5) and treated
with DNase I (500 units) at 4 °C for 5 min. The proteins in the
solution were precipitated by the addition of cold acetone to a final
concentration of 80%. The solution was kept at 0 °C for 15 min and
centrifuged at 3000 rpm for 20 min. The precipitates were extracted
successively three times with acetone/water (4:1), three times with
chloroform/methanol (2:1), four times with chloroform/methanol/water
(10:10:3), and once with ethanol/water (4:1). The delipidated proteins
were dispersed in ethanol/water (4:1) and stored at
20 °C before
use. The protein concentration was estimated by the Bio-Rad protein
assay method with bovine plasma
-globulin as a standard.
Analysis of Delipidated Proteins--
Delipidated proteins were
subjected to 12 or 7.5% SDS-PAGE and visualized by CBB or PAS stain.
The PAS staining was performed according to a procedure described
previously (10). Radioactive proteins on the gel were detected by
autoradiography with Fuji RX x-ray film after fluorometric enhancement
by EN3HANCE (NEN Life Science Products).
Analysis of Trypsin-digested Peptides--
The delipidated
proteins obtained from cells labeled with [3H]GOH or
[3H]GGPP were dissolved in a solution containing 10 mM MgCl2, 1 mM dithiothreitol, and
100 mM Tris-HCl buffer (pH 8.0) and digested with 0.75 mg/ml trypsin at 37 °C for 12 h. The trypsin-digested peptides
were subjected to 12% SDS-PAGE, and the gel corresponding to
radioactive peptides was cut off and extracted with water at 37 °C.
The recovered peptides were treated with methyl iodide as described
(5). They were also incubated with 0.5 M HCl for 2 h
at 37 or 95 °C and extracted with chloroform/methanol (2:1). The
radioactive extracts obtained by strong acid treatment were evaporated
under a stream of N2 and analyzed by normal-phase TLC (Merck Kieselgel 60) in a solvent system of
1-propanol/NH4OH/H2O (6:3:1). The radioactivity
on the plate was detected with a Fuji BAS1000 bioimaging analyzer. The
silica gel corresponding to radioactive materials was scraped from the
TLC plate and extracted with chloroform/methanol (2:1). The extracts
were evaporated under a stream of N2. Enzymatic dephosphorylation of the extracts was performed as described previously (11). Briefly, the sample was suspended in 20 µl of ethanol, and to
the suspension was added 0.1 mg of wheat germ acid phosphatase containing 200 µl of 50 mM Tris-maleate (pH 6.2) and
0.5% (w/v) octyl
-D-glycoside. The mixture was
incubated at 37 °C for 6 h, and the hydrolysates were extracted
with pentane. The extracts were evaporated under a stream of
N2 and analyzed by normal-phase TLC (Kieselgel 60) or
reverse-phase C18 TLC (Whatman LKC18) in a
solvent system of hexane/acetone (20:1, double development) or
acetone/water (40:1), respectively. The positions of standard compounds
were detected by exposing the plates to iodine vapor or by spraying the
plates with 10% (w/v) phosphomolybdate/methanol solution. The plates
were cut into 0.5-cm sections along the developed line, and the
radioactivity of the gels corresponding to each section was measured by
liquid scintillation counting.
In addition, delipidated proteins of H. halobium cells were
separated by 12% SDS-PAGE after trypsin digestion, electrotransferred to a polyvinylidene difluoride membrane, and PAS-stained. The membrane
corresponding to the PAS stain-positive band (28 kDa) was excised and
subjected to automated Edman degradation.
Analysis by Mass Spectrometry--
A sufficient amount of the
trypsin-digested peptides was subjected to 12% SDS-PAGE, and the gel
corresponding to 28-kDa peptide bands was cut off and extracted with
water at 37 °C. The recovered peptides were mixed with radioactive
28-kDa peptides. The radioactive materials released by acid treatment
of the mixed peptides were purified by reverse-phase C18
HPLC (YMC, 250 × 4.6 mm) according to a modified method as
described previously (12). Briefly, the radioactive materials were
eluted from the column maintained at 45 °C using a solvent system of
methanol/isopropyl alcohol (the isopropyl alcohol contained 10 mM phosphoric acid) (95:5). A pre-run was necessary to
ensure reproducible results. The radioactive fractions were combined
and washed with water to remove phosphoric acid. The purified materials
were treated with acid phosphatase, and the hydrolysates were further
purified by normal-phase HPLC (TOSOH, 250 × 4.6 mm) in a solvent
system of hexane/acetone (50:1). Mass spectra of the purified
hydrolysates were recorded on a Jeol JMS-DX600 mass spectrometer
(laboratory of Dr. M. Kira, Tohoku University). The potential of the
ionizing beam was 70 eV.
 |
RESULTS |
Previous work from this laboratory showed that
[3H]mevalonic acid was incorporated into H. halobium cells and that several proteins with molecular masses
between 35 and 45 kDa were predominantly radiolabeled (5). It was also
shown that 19-23% of the total radioactivity in the delipidated
proteins was released in the sulfonium salt cleavage reaction with
methyl iodide and that a diphytanylglyceryl group was associated with
proteins in thioether linkage (6). In the course of the current
experiments, we became aware of the occasional appearance of
radioactive protein bands with molecular masses <35-45 kDa.
Halobacteria are known to produce extracellular protease (13), and
Mescher et al. (14) have already reported that isolated
halobacterial cell-surface glycoprotein preparations contain a protease
activity. Therefore, we tested the action of endogenous protease during
protein preparation because we have used incubation conditions
(37 °C, >10 min) that reduce the viscosity of the cell lysis
solution by DNase I treatment. As shown Fig.
1A, the high molecular mass
protein bands were reduced with increasing incubation times. Fig.
1B shows CBB-stained protein bands of
[3H]mevalonic acid-labeled delipidated protein fractions
after 5 min of incubation at 4 °C (lane a) and after >10
min of incubation at 37 °C (lane b), described in a
previous report (5, 6). The fraction in lane b contained
partly degraded proteins. When the same fractions as in Fig.
1B (lanes a and b) were treated with
methyl iodide, ~23% of the total radioactivity of the latter was, in
fact, cleavable, but that of the former was only 3% released.

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Fig. 1.
Protein degradation by endogenous
protease. A, the cell pellets were lysed in 10 mM Tris-HCl buffer (pH 7.5) and treated with DNase I at
37 °C for 1, 10, 100, and 720 min. The proteins in the solution were
precipitated by the addition of ethanol to a final concentration of
80%, subjected to 12% SDS-PAGE, and visualized with CBB.
B, shown are the results from 12% SDS-PAGE of
[3H]mevalonic acid-labeled delipidated proteins after 5 min of incubation at 4 °C (lane a) and after >10 min of
incubation at 37 °C (lane b) with DNase I as described
previously (5, 6). The protein bands were visualized with CBB.
Molecular mass protein markers are shown in the last lane in
A and in the first lane in B.
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To characterize halobacterial isoprenoid-modified proteins, we again
labeled the cells with several radioactive tracers such as
[1-3H]GOH, [1-3H]FOH,
[1-3H]GGOH, [1-3H]GGP,
[1-3H]GGPP, and
(R)-[5-3H]mevalonic acid. After labeling with
each radioactive tracer (0.2 µM) for 96 h,
delipidated proteins were prepared without degradation. Fig.
2 (A and B) shows
CBB-stained protein bands and radioactive bands on SDS-PAGE of each
delipidated protein fraction, respectively. Incorporation of the
radioactive isoprenoid tracers into delipidated proteins was greater
than that of [3H]mevalonic acid. The major radioactive
band was detected in the region corresponding to 200 kDa, except in the
case of [3H]GGOH labeling. Since H. halobium
is known to have a characteristic cell-surface glycoprotein with a
molecular mass of 200 kDa (14, 15), we next tried to perform 7.5%
SDS-PAGE of each delipidated protein fraction to see the relation
between the major radioactive protein and the PAS-stainable
glycoprotein. As shown in Fig.
3A, the PAS-stained
glycoprotein bands were observed as reported (14), and the major
radioactive band (200 kDa) in each sample was in accord with the
PAS-stained glycoprotein band (200 kDa) (Fig. 3B). When the
[3H]mevalonic acid-, [3H]GOH-, and
[3H]GGPP-labeled delipidated proteins were treated with
methyl iodide, the radioactivity yields into an organic solvent were
<3%. These results suggest that the diphytanylglycerylated proteins
reported in previous studies (5, 6) are the minor components among isoprenoid-modified proteins in this halobacterium and that the major
component is the PAS stain-positive cell-surface glycoprotein. To
examine whether the radioactive lipid in the 200-kDa protein is
covalently bound to the known 200-kDa glycoprotein,
[3H]GOH-labeled delipidated proteins were treated with
trypsin and analyzed by 12% SDS-PAGE. As shown in Fig.
4A, no protein bands stained
with CBB were detected after trypsin digestion. However, a broad
radioactive band consisting of two materials (~28 kDa) was detected
by autoradiography of the same gel (Fig. 4B). The two
radioactive materials were also PAS-stained (Fig. 4C).
Almost all the PAS-stained saccharides of the 200-kDa proteins were
recovered in those of the two 28-kDa materials after trypsin digestion. To further examine whether the 28-kDa materials contain peptides, we
treated radioactive delipidated proteins with Pronase. No radioactive PAS-stained 28-kDa materials were observed by SDS-PAGE (data not shown), indicating that the 28-kDa materials are isoprenoid-modified glycopeptides.

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Fig. 2.
12% SDS-PAGE of delipidated proteins.
Cells were grown in the presence of 5 µCi/ml of
[3H]mevalonic acid (lane a),
[3H]GOH (lane b), [3H]FOH
(lane c), [3H]GGOH (lane d),
[3H]GGP (lane e), or [3H]GGPP
(lane f) for 96 h at 38 °C. A,
CBB-stained delipidated proteins; B, fluorograms of the delipidated
proteins shown in A. Each delipidated protein fraction was
loaded with the same amount of proteins. The x-ray film was exposed to
the gel at 80 °C for 60 days. Molecular mass protein markers are
shown in the first lane in A.
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Fig. 3.
7.5% SDS-PAGE of delipidated proteins.
Cells were labeled with [3H]mevalonic acid (lane
a), [3H]GOH (lane b),
[3H]FOH (lane c), [3H]GGOH
(lane d), [3H]GGP (lane e), or
[3H]GGPP (lane f) as described in the legend
of Fig. 2. A, PAS-stained delipidated proteins; B,
fluorograms of the delipidated proteins shown in A. Each
delipidated protein fraction was loaded with the same amount of
proteins. The x-ray film was exposed to the gel at 80 °C for 60 days. The arrow indicates the position of a 200-kDa protein
marker.
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Fig. 4.
12% SDS-PAGE of trypsin-digested delipidated
proteins. [3H]GOH-labeled delipidated proteins were
incubated in the absence (lane a) or presence (lane
b) of trypsin. A, CBB-stained delipidated proteins;
B, fluorograms of the delipidated proteins shown in
A; C, PAS-stained delipidated proteins. The x-ray
film was exposed to the gel at 80 °C for 30 days. Molecular mass
protein markers are shown in the first lane in
A.
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To obtain insight into the nature of the isoprenoid binding manner,
radioactive PAS-stained peptides (28 kDa) recovered from the gel were
treated with methyl iodide for 24 h, with 0.5 M HCl for 2 h at 37 °C, or with 0.5 M HCl for 2 h at
95 °C. No radioactive materials were released from the peptides in
the sulfonium salt cleavage reaction or under the acidic conditions at
37 °C, but under the acidic conditions at 95 °C, radioactive
materials were released from the peptides with 90% recovery. The
released materials remained at the origin on a normal-phase TLC plate
when a non-polar solvent of benzene/ethyl acetate (9:1) was used, but
showed a mobility similar to that of dolichyl (C55)
phosphate under the chromatographic conditions using a polar solvent
(Fig. 5). From the chromatographic
behavior and chemical properties of the released materials, we
speculated that they were non-allylic phosphate ester compounds, such
as dolichyl (C60) phosphate (16) or diphytanylglyceryl phosphate (17). When the radioactive materials recovered from the TLC
plate were treated with acid phosphatase, ~30-40% of the radioactivity of the polar materials was actually detected in pentane-soluble hydrolysates. As shown in Fig.
6, the radioactive hydrolysates moved
with a spot of diphytanylglycerol, not with those of dolichol
(C55-65), on normal- and reverse-phase C18 TLC
plates. We also performed normal- or reverse-phase TLC of the
radioactive hydrolysates in another solvent system of hexane/ether (95:5) or acetone/methanol (40:1). The hydrolysates co-migrated with
diphytanylglycerol on the TLC plates (data not shown). To confirm the
structure of the hydrolysates by mass spectrometry, we prepared a
sufficient amount of the radioactive materials released by acid
treatment of trypsin-digested peptides and purified them by
reverse-phase C18 HPLC. After dephosphorylation with acid
phosphatase (40-50% yield), the hydrolysates were further purified by
silica gel HPLC. As shown in Fig. 7, the
mass spectrum was identical to that of diphytanylglycerol with respect
to a parent ion at m/z 652 (C43H88O3) and its subsequent
fragmentation. These results indicate that the 28-kDa glycopeptides are
modified with diphytanylglyceryl phosphate.

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Fig. 5.
Normal-phase TLC of the materials released by
acid treatment of the radioactive PAS-stainable peptides. The
radioactive PAS-stainable peptides (28 kDa) described under
"Experimental Procedures" were incubated with 0.5 M HCl
for 2 h at 95 °C. The released materials were mixed with
dolichyl (C55) monophosphate and analyzed by normal-phase
TLC in a solvent system of 1-propanol/NH4OH/H2O
(6:3:1). Dolichyl monophosphate was detected with iodine vapor
(lane A). The radioactivity on the plate was detected with a
Fuji BAS1000 bioimaging analyzer (lane B). The
arrow indicates the position of dolichyl (C55)
phosphate (Dol-P). Ori., origin; S. F., solvent front.
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Fig. 6.
Enzymatic dephosphorylation of the materials
released by acid treatment of the radioactive peptides. The
radioactive PAS-stainable peptides (28 kDa) described under
"Experimental Procedures" were incubated in the presence or absence
of acid phosphatase. The hydrolysates were analyzed by normal-phase TLC
in a solvent system of hexane/acetone (20:1, double development)
(A) and by reverse-phase C18 TLC in a solvent
system of acetone/H2O (40:1) (B). Arrows
a-d indicate the positions of authentic standards
(C55 dolichol, C60 dolichol , C65
dolichol, and diphytanylglycerol, respectively). Ori.,
origin; S. F., solvent front.
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Fig. 7.
Mass spectrograms. The radioactive
materials released by acid treatment of the PAS-stainable peptides (28 kDa) were purified by reverse-phase C18 HPLC. The purified
materials were dephosphorylated by acid phosphatase treatment, and the
hydrolysates were further purified by normal-phase HPLC as described
under "Experimental Procedures." A and B,
mass spectrograms of the purified hydrolysate and the
diphytanylglycerol standard, respectively.
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To confirm that the diphytanylglyceryl phosphate-modified glycopeptides
are in fact derived from the known cell-surface glycoproteins (200 kDa), we tried to determine the N-terminal amino acid sequences of the
peptides. The delipidated proteins from halobacterial cells were
digested with trypsin and subjected to 12% SDS-PAGE, followed by
electrotransfer to a polyvinylidene difluoride membrane. The PAS
stain-positive bands (28 kDa) overlapped. The membrane corresponding to
the overlapped band was excised and subjected to automated Edman
degradation. The peptides were analyzed and shown to be a mixture of
two peptides with the N-terminal sequences QNVEIVEELEEP (peptide I) and
NVEIVEELEEPD (peptide II) in a ratio of 1:2. As shown in Fig.
8A, peptide I corresponded to
a C-terminal fragment (amino acids 731-816) of the halobacterial
cell-surface glycoprotein (18). Peptide II also corresponded to this
fragment, but lacked the first N-terminal amino acid, glutamine. These
results indicate that diphytanylglyceryl phosphate is covalently
associated with the C-terminal region of the cell-surface
glycoprotein.

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Fig. 8.
Schematic representation of the cell-surface
glycoprotein of H. halobium (A) and
comparison of protein sequences from the COOH termini of the
cell-surface glycoproteins of H. halobium
(1), H. volcanii
(2), and H. japonica
(3) (B).
Numbering indicates amino acid positions. M,
membrane-binding domain; T, region of a threonine cluster;
, the repeated saccharide unit; , N-linked
oligosaccharides; , O-linked disaccharides. I
and II indicate partial sequences analyzed by Edman
degradation. Three repeats of the amino acid sequence motif
(D/E)(T/S)5 and one repeat of the motif TTXEPTE
in H. volcanii (2) are indicated by
dashed and solid underlining, respectively. Six
repeats of the amino acid sequence motif
PXTXTXE in H. japonica
(3) are indicated by underlining. For further
explanations, see "Discussion."
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DISCUSSION |
This study has demonstrated that the known cell-surface
glycoprotein of H. halobium is modified with
diphytanylglyceryl phosphate. This protein has already been well
characterized as the first prokaryotic glycoprotein consisting of 818 amino acids (18), as shown in Fig. 8A. A single high
molecular mass saccharide that is composed of repeats of sulfated
pentasaccharides is associated with an asparagine residue (position 2).
Ten sulfated (HexUA1-4)2-3Glc oligosaccharides are
connected to asparagine residues (positions 305, 364, 404, 479, 609, 693, 717, 753, 777, and 781). About 20 Glu1-2Gal disaccharides are
O-glycosidically attached to threonine residues, of which 14 threonine residues form a cluster (positions 755-774) adjacent to a
stretch of 21 amino acids (positions 795-815) exclusively composed of
hydrophobic residues (membrane-binding domain). The highly
N- and O-glycosidically modified protein behaves as if it had an apparent molecular mass of 200 kDa on SDS-PAGE. We
showed that a PAS stain-positive 200-kDa protein was radioactive when
several tritium-labeled isoprenoid compounds (except for [3H]GGOH) were used as tracers. We could not readily
believe in the covalent isoprenoid modification of the glycoprotein
because this glycoprotein has been characterized in detail (described above). However, when the trypsin-digested PAS-stained peptides were
found to be radioactive, we believed in a modification of the known
cell-surface glycoprotein by isoprenoid-derived materials.
Although it is expected that all the saccharides on the 200-kDa protein
can be stained with PAS, the majority of the PAS-stained saccharides
were recovered in the two trypsin-digested peptides (28 kDa), which
corresponded to a C-terminal fragment containing a threonine cluster.
The O-linked saccharides might be predominantly stained
because sulfated N-linked saccharides may meet with
repulsion by negatively charged periodate ions (19). As shown in Fig. 4, the two 28-kDa peptides were apparently radioactive and PAS stain-positive. The mobility difference (~1 kDa) between the two peptides might be due to the degree of glycosylation (20).
Electrotransfer of the peptides to a polyvinylidene difluoride membrane
also gave two PAS-stained bands, but the bands overlapped each other.
The peptides corresponding to the overlapped band were analyzed by Edman degradation and determined to be a mixture of two components. The
sequence of the minor component was exactly the same as the N-terminal
partial sequence of a fragment of a cell-surface glycoprotein, whereas
that of the major one lacked the N-terminal glutamine residue. Since
trypsin cleaves peptide bonds involving the carboxyl groups of arginine
and lysine, the major peptide fragment lacking glutamine implies the
occurrence of an isoform that contains either an RRNVEIVE
or RKNVEIVE sequence different from the
RQNVEIVE sequence found in the known cell-surface glycoprotein.
The linkage between unknown lipids and peptides of the 28-kDa
trypsin-digested fragments was resistant to the sulfonium salt cleavage
reaction, but was cleavable by acid at 95 °C, yielding non-allylic
phosphate ester lipids. Neither phosphate ester nor pyrophosphate ester
lipids were released under the mild acidic conditions, suggesting the
presence of a phosphodiester linkage (21). This result led us to
speculate that the lipid might be dolichyl (C60) phosphate
because dolichyl phosphate modification of proteins has been already
reported in mammalian liver (22), and the carbon chain length of
dolichyl phosphate occurring in H. halobium is
C60 (16). However, the radioactive hydrolysates released by
acid phosphatase treatment co-migrated with diphytanylglycerol on
thin-layer chromatography, and the HPLC-purified hydrolysate was
identified to be diphytanylglycerol by mass spectrometry. Although the
exact type of linkage of diphytanylglycerol to the proteins remains to
be established, at present, we conclude that the non-allylic lipid is
diphytanylglyceryl phosphate from its chromatographic and chemical
properties. Coupled with previous findings (5, 6), these results
indicate that H. halobium contains two new types of
isoprenoid-modified proteins, namely diphytanylglycerylated proteins
with thioether linkage and diphytanylglyceryl phosphate-modified
proteins. Two similar types of isoprenoid modification, dolichyl
proteins with thioether linkage and dolichyl phosphate-modified proteins, have also been reported in mammalian tissues (22, 23).
The cell-surface glycoproteins of H. halobium,
Haloferax volcanii, and Haloarcula japonica have
been reported to show a considerable homology in amino acid sequences
(18, 24, 25). Fig. 8B shows the protein sequences from the
COOH termini of the three cell-surface glycoproteins. The C-terminal
membrane-binding domain is conserved in these glycoproteins. However,
the cluster of O-glycosylated threonine residues found in
H. halobium is not necessarily conserved. In H. volcanii, the threonine cluster is much longer and contains three
repeats of the amino acid sequence motif (D/E)(T/S)5 and a
repeat of the motif TTXEPTE. Six repeats of the amino acid
sequence motif PXTXTXE are present in
H. japonica instead of the threonine-rich cluster. Kessel
et al. (26) have speculated that the unusual structural
threonine-rich cluster element serves as a spacer between the
membrane-binding domain and the extracellular domain of the cell-surface glycoprotein, thus creating an interspace that may be
regarded as analogous to the periplasmic space of Gram-negative eubacteria. Therefore, if the modification of the C-terminal domain by
diphytanylglyceryl phosphate is common to these archaebacterial glycoproteins and if the hydrophobic diphytanylglyceryl group also
serves as another membrane anchor, the modification site might be in a
limited region adjacent to hydrophobic residues corresponding to the
membrane-binding domain. We speculate that the hydroxyl group of the
side chain of serine or threonine next to each membrane-binding domain
might be a possible modification site. However, if the modification by
diphytanylglyceryl phosphate is characteristic of H. halobium, the lipid would be associated with a hydroxyl group of
either saccharides or the other peptide residues.
Kobayashi et al. (27) have recently reported the presence of
glycosylphosphatidylinositol-anchored proteins even in an
archaebacterium, Sulfolobus acidocaldarius. Since inositol
has not been found in halobacteria (28), the possibility that
glycosylphosphatidylinositol-anchored proteins occur in this
archaebacterium would be ruled out. In a preliminary experiment, no
radioactivity was, in fact, detected in delipidated protein fractions
obtained by metabolic labeling with 14C-labeled
ethanolamine or inositol (data not shown). The C-terminal hydrophobic
peptide chain of the cell-surface glycoprotein is followed by three
basic amino acids, as shown in Fig. 8B, indicating that the
hydrophobic peptide is not removed (29). Therefore, it is thought that
the surface glycoprotein of H. halobium is a novel protein
that has not only one single C-terminal hydrophobic peptide chain, but
also another hydrophobic lipid chain, unlike mature
glycosylphosphatidylinositol-anchored proteins free from the C-terminal
hydrophobic peptide of their precursor proteins.
Halophilic archaea lack the peptidoglycan layer, and the cell-surface
glycoprotein is the only cell wall component (30). The glycoproteins of
H. halobium, H. volcanii, and H. japonica are essential for maintaining the rod shape, the flat
disc shape, and the triangular disc shape, respectively (31, 32). Each mature polypeptide contains 818, 794, and 828 amino acids with molecular masses of 86,538, 81,732, and 87,166 Da. These values are
much lower than those estimated from SDS-PAGE (200, 170, and 170 kDa,
respectively). The same abnormal electrophoretic behavior was found for
the deglycosylated surface-layer polypeptide of H. halobium
(18). Therefore, these polypeptides may have reduced capacity for SDS
binding due to their unusual content of acidic residues such as
aspartic acid and glutamic acid (18, 24). In this study, we presented a
novel diphytanylglyceryl phosphate modification of the halobacterial
cell-surface glycoprotein. This modification seems to be common to the
glycoproteins of halophilic archaebacteria and might partly cause the
reduced electrophoretic mobility of the glycoproteins on SDS-PAGE.
Further experiments are in progress to identify the isoprenoid
modification site of the cell-surface glycoprotein of H. halobium.
 |
ACKNOWLEDGEMENTS |
We thank T. Ishima and I. Kon for the
measurement of mass spectrograms.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid from the
Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-22-217-5622;
Fax: 81-22-217-5620; E-mail: yasagami{at}icrs.tohoku.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
GOH, geraniol;
FOH, farnesol;
GGOH, geranylgeraniol;
GGP, geranylgeranyl monophosphate;
GGPP, geranylgeranyl diphosphate;
PAGE, polyacrylamide gel
electrophoresis;
CBB, Coomassie Brilliant Blue;
PAS, periodic
acid-Schiff;
HPLC, high performance liquid chromatography.
 |
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