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• W2007528488 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. 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. 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 (1Clarke S. Annu. Rev. Biochem. 1992; 61: 355-386Crossref PubMed Scopus (793) Google Scholar, 2Marshall C.J. Science. 1993; 259: 1865-1866Crossref PubMed Scopus (303) Google Scholar, 3Casey P.J. Curr. Opin. Cell Biol. 1994; 6: 219-225Crossref PubMed Scopus (169) Google Scholar). 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 (4Epstein W.W. Lever D. Leining L.M. Bruenger E. Rilling H.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9668-9670Crossref PubMed Scopus (123) Google Scholar). 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar, 6Sagami H. Kikuchi A. Ogura K. J. Biol. Chem. 1995; 270: 14851-14854Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). 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. (R)-[5-3H]Mevalonic acid, [1-3H]GOH, 1The 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. [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 (7Danilov L.L. Chojnacki T. FEBS Lett. 1981; 131: 310-312Crossref Scopus (75) Google Scholar). 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 Minnikinet al. (8Minnikin D.E. Alshamaony L. Goodfellow M. J. Gen. Microbiol. 1975; 88: 200-204Crossref PubMed Scopus (407) Google Scholar) and confirmed by mass spectrometry (9Kushwaha S.C. Kramer J.K.G. Kates M. Biochim. Biophys. Acta. 1975; 398: 303-314Crossref PubMed Scopus (57) Google Scholar). Trypsin (code no. 109819) and Pronase (code no. 165921) were purchased from Roche Molecular Biochemicals. All other chemicals were from commercially available sources. 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. 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 γ-globulin as a standard. 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 (10Zacharius R.M. Zell T.E. Morrison J.H. Woodlock J.J. Anal. Biochem. 1969; 30: 148-152Crossref PubMed Scopus (1679) Google Scholar). Radioactive proteins on the gel were detected by autoradiography with Fuji RX x-ray film after fluorometric enhancement by EN3HANCE (NEN Life Science Products). 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar). 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 (11Carson D.D. Lennarz W.J. J. Biol. Chem. 1981; 256: 4679-4686Abstract Full Text PDF PubMed Google Scholar). 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 β-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. 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 C18HPLC (YMC, 250 × 4.6 mm) according to a modified method as described previously (12Chaudhary N. Freeman D.J. Rip J.W. Carroll K.K. Lipids. 1982; 17: 558-560Crossref PubMed Scopus (26) Google Scholar). 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. 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar). 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 (6Sagami H. Kikuchi A. Ogura K. J. Biol. Chem. 1995; 270: 14851-14854Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). 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 (13Norberg P. Hofsten B.V. J. Gen. Microbiol. 1969; 55: 251-256Crossref PubMed Scopus (70) Google Scholar), and Mescher et al. (14Mescher M.F. Strominger J.L. Watson S.W. J. Bacteriol. 1974; 120: 945-954Crossref PubMed Google Scholar) 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.1 A, the high molecular mass protein bands were reduced with increasing incubation times. Fig.1 B 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar, 6Sagami H. Kikuchi A. Ogura K. J. Biol. Chem. 1995; 270: 14851-14854Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The fraction in lane b contained partly degraded proteins. When the same fractions as in Fig.1 B (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. 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. halobiumis known to have a characteristic cell-surface glycoprotein with a molecular mass of 200 kDa (14Mescher M.F. Strominger J.L. Watson S.W. J. Bacteriol. 1974; 120: 945-954Crossref PubMed Google Scholar, 15Mescher M.F. Strominger J.L. J. Biol. Chem. 1976; 251: 2005-2014Abstract Full Text PDF PubMed Google Scholar), 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.3 A, the PAS-stained glycoprotein bands were observed as reported (14Mescher M.F. Strominger J.L. Watson S.W. J. Bacteriol. 1974; 120: 945-954Crossref PubMed Google Scholar), and the major radioactive band (200 kDa) in each sample was in accord with the PAS-stained glycoprotein band (200 kDa) (Fig. 3 B). 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar, 6Sagami H. Kikuchi A. Ogura K. J. Biol. Chem. 1995; 270: 14851-14854Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) 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.4 A, 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. 4 B). The two radioactive materials were also PAS-stained (Fig. 4 C). 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.Figure 37.5% 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.View Large Image Figure ViewerDownload (PPT)Figure 412% 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 inA; 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 inA.View Large Image Figure ViewerDownload (PPT) 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 (16Lechner J. Wieland F. Sumper M. J. Biol. Chem. 1985; 260: 860-866Abstract Full Text PDF PubMed Google Scholar) or diphytanylglyceryl phosphate (17Moldoveanu N. Kates M. Biochim. Biophys. Acta. 1988; 960: 164-182Crossref Scopus (41) Google Scholar). 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.Figure 6Enzymatic 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 , C65dolichol, and diphytanylglycerol, respectively). Ori., origin; S. F., solvent front.View Large Image Figure ViewerDownload (PPT)Figure 7Mass 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.View Large Image Figure ViewerDownload (PPT) 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.8 A, peptide I corresponded to a C-terminal fragment (amino acids 731–816) of the halobacterial cell-surface glycoprotein (18Lechner J. Sumper M. J. Biol. Chem. 1987; 262: 9724-9729Abstract Full Text PDF PubMed Google Scholar). 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. 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 (18Lechner J. Sumper M. J. Biol. Chem. 1987; 262: 9724-9729Abstract Full Text PDF PubMed Google Scholar), as shown in Fig. 8 A. 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 areO-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 highlyN- 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 (19Scott J.E. Harbinson R.J. Histochemie. 1968; 14: 215-220Crossref PubMed Scopus (65) Google Scholar). 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 (20Mescher M.F. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2687-2691Crossref PubMed Scopus (88) Google Scholar). 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 (21Warren C.D. Jeanloz R.W. Methods Enzymol. 1978; 50: 122-137Crossref PubMed Scopus (30) Google Scholar). 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 (22Thelin A. Löw P. Chojnacki T. Dallner G. Eur. J. Biochem. 1991; 195: 755-761Crossref PubMed Scopus (27) Google Scholar), and the carbon chain length of dolichyl phosphate occurring in H. halobium is C60 (16Lechner J. Wieland F. Sumper M. J. Biol. Chem. 1985; 260: 860-866Abstract Full Text PDF PubMed Google Scholar). 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 (5Sagami H. Kikuchi A. Ogura K. Fushihara K. Nishino T. Biochem. Biophys. Res. Commun. 1994; 203: 972-978Crossref PubMed Scopus (6) Google Scholar, 6Sagami H. Kikuchi A. Ogura K. J. Biol. Chem. 1995; 270: 14851-14854Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), 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 (22Thelin A. Löw P. Chojnacki T. Dallner G. Eur. J. Biochem. 1991; 195: 755-761Crossref PubMed Scopus (27) Google Scholar, 23Hjertman M. Wejde J. Dricu A. Carlberg M. Griffiths W.J. Sjövall J. Larsson O. FEBS Lett. 1997; 416: 235-238Crossref PubMed Scopus (28) Google Scholar). The cell-surface glycoproteins of H. halobium,Haloferax volcanii, and Haloarcula japonica have been reported to show a considerable homology in amino acid sequences (18Lechner J. Sumper M. J. Biol. Chem. 1987; 262: 9724-9729Abstract Full Text PDF PubMed Google Scholar, 24Sumper M. Berg E. Mengele R. Strobel I. J. Bacteriol. 1990; 172: 7111-7118Crossref PubMed Scopus (173) Google Scholar, 25Wakai H. Nakamura S. Kawasaki H. Takada K. Mizutani S. Aono R. Horikoshi K. Extremophiles. 1997; 1: 29-35Crossref PubMed Scopus (24) Google Scholar). Fig. 8 B 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 inH. 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 inH. japonica instead of the threonine-rich cluster. Kesselet al. (26Kessel M. Wildhaber I. Cohen S. Baumeister W. EMBO J. 1988; 7: 1549-1554Crossref PubMed Google Scholar) 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. (27Kobayashi T. Nishizaki R. Ikezawa H. Biochim. Biophys. Acta. 1997; 1334: 1-4Crossref PubMed Scopus (40) Google Scholar) have recently reported the presence of glycosylphosphatidylinositol-anchored proteins even in an archaebacterium, Sulfolobus acidocaldarius. Since inositol has not been found in halobacteria (28Koga Y. Nishihara M. Morii H. Akagawa-Matsushita M. Microbiol. Rev. 1993; 57: 164-182Crossref PubMed Google Scholar), 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. 8 B, indicating that the hydrophobic peptide is not removed (29Ferguson M.A.J. Williams A.F. Annu. Rev. Biochem. 1988; 57: 285-320Crossref PubMed Scopus (952) Google Scholar). 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 (30Mescher M.F. Strominger J.L. Prog. Clin. Biol. Res. 1977; 17: 459-465PubMed Google Scholar). The glycoproteins ofH. halobium, H. volcanii, and H. japonica are essential for maintaining the rod shape, the flat disc shape, and the triangular disc shape, respectively (31Baumeister W. Lembcke G. J. Bioenerg. Biomembr. 1992; 24: 567-575Crossref PubMed Scopus (107) Google Scholar, 32Horikoshi K. Aono R. Nakamura S. Experientia (Basel). 1993; 49: 497-502Crossref Scopus (26) Google Scholar). 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(18Lechner J. Sumper M. J. Biol. Chem. 1987; 262: 9724-9729Abstract Full Text PDF PubMed Google Scholar). 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 (18Lechner J. Sumper M. J. Biol. Chem. 1987; 262: 9724-9729Abstract Full Text PDF PubMed Google Scholar, 24Sumper M. Berg E. Mengele R. Strobel I. J. Bacteriol. 1990; 172: 7111-7118Crossref PubMed Scopus (173) Google Scholar). 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. We thank T. Ishima and I. Kon for the measurement of mass spectrograms." @default.
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