Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, 352 Sakaecho, Itabashi-ku, Tokyo 1730015, Japan, 2KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1135 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 2360004, Japan, 3Research Department, The Noguchi Institute, 181 Kaga, Itabashi-ku, Tokyo 1730003, Japan, and 4Department of Biochemistry, Institute of Medical Science, University of Tokyo, 461 Shirokanedai, Minato-ku, Tokyo 1088639, Japan
Received on June 1, 2000; revised on September 11, 2000; accepted on September 12, 2000.
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Abstract |
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Key words: GlcNAc transferase/GlcNAc1-2/GnT-I/GnT-II/O-mannosyl glycan
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Introduction |
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O-Mannosylation is known as a yeast-type modification (Nakajima and Ballou, 1974; Raizada et al., 1975
; Hayette et al., 1992
; Herscovics and Orlean, 1993
), and oligomannose-type O-mannosylated glycoproteins are abundant in the yeast cell wall. Other than that in fungi, O-linked GlcA
1-6Man in the skin collagen of clam worm has been reported (Spiro and Bhoyroo, 1980
). On the other hand, among mammalian proteins, several glycoproteins were modified by a similar type of glycosylation (Endo, 1999
). Interestingly, a series of mammalian type O-mannosyl glycans, with heterogeneous Man-branching (2- or 3-substituted and 2,6-disubstituted mannoses) and peripheral structures (Sia
2-3Gal
1-4GlcNAc
1-2Man, Gal
1-4GlcNAc
1-2Man, Gal
1-4(Fuc
1-3)GlcNAc
1-2Man, HSO3-3GlcA
1-3Gal
1-4GlcNAc
1-2Man) have been found (Finne et al., 1979
; Wing et al., 1992
; Chiba et al., 1997
; Yuen et al., 1997
; Sasaki et al., 1998
; Smalheiser et al., 1998
; Chai et al., 1999
). We demonstrated that sialyl mannosyl glycan was one of laminin-binding ligands of
-dystroglycan (Yamada et al., 1996
; Chiba et al., 1997
), while the detailed biological significance of this type of glycosylation is not fully understood.
Analysis of the biosynthetic pathway of the O-mannosyl glycans in mammalian cells is important for elucidating not only the regulation of expression but also the biological functions of these glycans. To our knowledge, there are no reports concerning the O-mannosylation pathway in mammals.
In this report, we describe the development of an enzyme assay for UDP-N-acetylglucosamine: O-linked mannose -1,2-N-acetylglucosaminyltransferase (hereafter referred to MGnT) and initial characterization of its activity. Its activity is probably different from UDP-N-acetylglucosamine:
-3-D-mannoside
-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine:
-6-D-mannoside
-1,2-N-acetylglucosaminyltransferase II (GnT-II), which is responsible for the formation of the GlcNAc
1-2Man linkage in the early steps of N-glycan processing (Schachter, 1994
). The identification and characterization of enzymes involved in the biosynthesis of mammalian type O-mannosyl glycans will be an important step forward elucidating these novel glycans.
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Results |
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In experiments using the new mannosyl peptide as an acceptor substrate, the activity of GlcNAc transfer to the mannosyl residue was detected in the neonatal rat brain extract (Figure 1). We tentatively named the GlcNAc transferase as MGnT. Although four peaks including a passed-through large peak were observed by the reversed-phase HPLC (Figure 1A), only the peak eluted at 30 min appeared dependent on the presence of mannosyl peptide (compare to Figure 1B). The remaining two peaks at approximately 21 min and 42 min were probably due to the incorporation of radioactive GlcNAc to unidentified endogenous substrates. The MGnT activity showed linear dependency on time, amount of protein, and amount of donor and acceptor substrates (Figure 2). This activity totally disappeared following heating at 100°C for 3 min (Figure 2B). The MGnT activity had an optimum pH between 7.0 and 7.5.
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Discussion |
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The dystroglycan complex links the extracellular matrix to the cytoskeleton in various tissues. -Dystroglycan, which is the cell surface component of the dystroglycan complex, is a heavily glycosylated protein, known to bind to laminin and agrin in the basal lamina of muscle cells and Schwann cells (Hemler, 1999
). Studies on targeted disruption of the mouse dystroglycan gene showed that dystroglycan is required for normal embryonic development beyond the egg cylinder stage (Williamson et al., 1997
) and formation of a basement membrane in embryoid bodies by binding soluble laminin and organizing it on the cell surface (Henry and Campbell, 1998
). In relation to these, we demonstrated that sialyl O-mannosyl glycan is one of the laminin-binding ligands of
-dystroglycan (Yamada et al., 1996
; Chiba et al., 1997
). In addition, two interesting studies on
-dystroglycan have been published recently (Cao et al., 1998
; Rambukkana et al., 1998
). Two unrelated pathogenic agents which are members of the arenavirus family, namely, the Lassa fever virus and Mycobacterium leprae (the bacterium responsible for leprosy), bind to target cells through interactions with a common receptor,
-dystroglycan. Quite interestingly, both results suggest the possible contribution of the glycan moieties of
-dystroglycan at the point of entry of these causative agents, although details of the underlying molecular events remain to be determined. Therefore, analysis of the biosynthetic pathway of the O-mannosyl glycans in mammalian cells will be important in elucidating not only the regulation of expression but also the biological functions of these glycans.
In the present study, we detect the 1,2-GlcNAc transferase activity with respect to the mannosyl residue on the peptide in several brains and two cell lines. It is of interest that this activity is not due to the action of conventional GnT-I and GnT-II, that are responsible for N-glycan processing. It has been shown that both GnT-I and GnT-II are very important for the processing. Gene knockout mice of GnT-I did not survive beyond day 10.5 post-fertilization and exhibited severe developmental abnormalities particularly in the brain (Ioffe and Stanley, 1994
; Metzler et al., 1994
). On the other hand, gene knockout mice of GnT-II survived to term, but were born stunted with various congenital abnormalities and died shortly after birth (Campbell et al., 1997
). Furthermore, children with a defective GnT-II gene showed severe psychomotor retardation and other multisystemic abnormalities (Charuk et al., 1995
; Tan et al., 1996
). These results indicate that the formation of GlcNAc
1-2Man of N-glycans is essential to cellcell interactions and morphogenesis of multicellular organisms. This step is necessary to generate diverse structures. Thus, the investigation of GlcNAc
1-2Man formation of mannosyl glycans will contribute to our knowledge of basic processes relevant to a wide range of physiological conditions in the future, although the details remains to be determined. It is noteworthy that although the presence of 2,6-disubstituted Man residue linked to GlcNAc residues has been reported in brain proteoglycans (Yuen et al., 1997
; Chai et al., 1999
), we could not detect the relevant activity under our experimental conditions. Furthermore, we also could not detect the
1,3-GlcNAc transferase activity with respect to the mannosyl residue on a synthetic peptide. The reason is not clear at this stage. The most possible explanation is that both the
1,3-GlcNAc transfer activity and the
1,6-GlcNAc transfer activity are too low to be detected under our assay conditions. Another possible explanation is that both enzymes may require the restriction peptide sequence or the specific location of Man on the protein. Further studies on purification and cloning of MGnT will be necessary to solve these problems. To our knowledge, there is only one report concerning the presence of the GlcNAc
1-3Man linkage in mammalian glycoproteins of brain proteoglycans (Finne et al., 1979
). A comment, however, must be made on the presence of Man substitution at C-3 in their study (Finne et al., 1979
). Mass spectrum data indicate that the substitution of Man was either at the C-2 position or C-3 position; based on fragment patterns of the trimethylsilyl derivatives of deuterated disaccharides, they concluded that a substitution of C-2 was unlikely (Finne et al., 1979
). However, a recent study suggests the occurrence of rearrangement during preparation of the sample involving the deuteration step (Yuen et al., 1997
). Therefore, it is important to confirm whether brain proteoglycans contain a Man substitution at C-3, because this is contrary to the finding of the predominance of Man substitution at C-2 in recent studies on glycoproteins including brain proteoglycans (Yuen et al., 1997
; Chai et al., 1999
).
O-Mannosyl-type linkages, Man-Ser/Thr, are known as a yeast-type modification (Nakajima and Ballou, 1974; Raizada et al., 1975
; Hayette et al., 1992
; Herscovics and Orlean, 1993
). Although the initial step of protein O-mannosylation in yeast is well studied, the pathway in mammals is still obscure and remains to be elucidated. In Saccharomyces cerevisiae, protein O-mannosylation is catalyzed by a family of protein mannosyltransferases encoded by seven genes (PMT1-7) (Strahl-Bosinger et al., 1999
). Recently, human (Pérez Jurado et al., 1999
) and rat (Chiba and Margolis, 1999
) homologs of the pmt gene were obtained. Furthermore, three GnT-I homologs (gly-12, gly-13, and gly-14) were cloned from Caenorhabditis elegans (Chen et al., 1999
). Interestingly, gly-12 and gly-14 showed enzyme activity but gly-13 did not. Therefore, it is of interest to examine whether or not such pmt homologs and gly-13 have protein O-mannosyltransferase activity and MGnT activity, respectively. These studies will pave the way forward elucidating the biosynthetic pathway of O-mannosyl glycans in mammalian cells.
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Materials and methods |
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Cell lines
Rat schwannoma cells, RT-4, were obtained from Dr. Matsumura (Teikyo University of School of Medicine) and cultured in DMEM (GIBCO BRL, Life Technologies, Inc., Rockville, MD) containing 10% fetal calf serum (FCS) in 10% CO2 at 37°C. Mouse myoblasts, C2C12, were obtained from Dr. Aoki (University of Tokyo) and cultured in DMEM containing 10% FCS in 5% CO2 at 37°C. After monolayer formation, myoblast cultures were induced to differentiate in DEME containing 2% horse serum and 0.5% FCS for 3 days.
The GnT-I-expressing S. cerevisiae strain (YPH500/pSY114; Yoshida et al., 1999) and the multicopy expression vector of human GnT-II (unpublished observations) for yeast were kindly provided by Dr. Satoshi Yoshida (KIRIN Brewery Co., Japan). The yeast strain was cultured and the lysates were used for the measurement of the activity of GnT-I and MGnT as described later. Construction of the S. cerevisiae strain which expresses GnT-II was performed as follows. The region coding a promoter, the entire GnT-II and a terminator was cut from the vector, and the fragment was ligated into an integration vector, pASZ10 (Stotz and Linder, 1990
). The plasmid was used for the transformation of yeast W3031A (Mata leu2-3, 112 his3-11, 15 ade2-1 ura3-1 trp1-1 can1-100). The transformant, WCY12, was cultured and the cell lysates were used for the measurement of the activities of the GnT-II and MGnT.
Synthesis of mannosyl peptide substrate
Mannosyl peptide (Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH2) was synthesized in a solid-phase manner using 9-fluorenyloxymethylcarbonyl (Fmoc) chemistry. Fmoc-amino acids were coupled by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety (Mizuno et al., 1998).
Fmoc-Thr(Man)-OH was synthesized as follows: The reaction of phenyl 2,3,4,6-tetra-O-benzyl-1-thio-D-mannopyranoside and N-benzyloxycarbonyl-L-threonine benzyl ester (Z-Thr-OBzl) in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave the desired protected mannosyl threonine derivative (Z-Thr(Man(OBzl)4)-OBzl) with 77% yield. After deprotection of all benzyl groups and Z group by catalytic hydrogenation, Fmoc-OSu was reacted with the residue to give the desired Fmoc-Thr(Man)-OH with 75% yield. The product was easily purified by solid-phase extraction using a polymeric adsorbent, such as Dianion HP-20 (Nippon Rensui Co., Tokyo, Japan) or Amberlite XAD-2 (Organo, Tokyo, Japan).
After the final deprotection from the glycopeptide resin, the crude mannosyl peptide was purified on a C18 preparative reversed-phase column (Inertsil ODS-3, 20 x 250 mm, GL Sciences Inc., Tokyo, Japan) eluted by mixing solvent A (0.1% trifluoroacetic acid (TFA) in distilled water) with solvent B (0.1% TFA in acetonitrile) at 45°C at a flow rate of 10 ml/min as follows: 25 min at 5% B; 10 min, linear gradient to 35% B. The glycopeptide separation was monitored continuously at 214 nm. The structure of the product was identified by 1H-NMR, amino acid analysis (6 M HCl, 110°C, 24 h) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results are as follows: amino acid analysis; Thr0.97Ala4.00Val1.07Pro3.20NH30.74; MALDI-TOF MS, Found: m/z[M+Na]+1020.11, calculated mass = [M+Na]+1020.10.
Preparation of rat brain extracts
Brains from new born rats (F344/N, Nihon SLC, Shizuoka, Japan) were homogenized on ice using a potter homogenizer (Iuchi Digital Homogenizer, Tokyo, Japan) containing 9 ml of 10 mM Tris-buffer (pH 7.4) with 1 mM EDTA and 250 mM sucrose with a protease inhibitor cocktail (2 µg/ml antipain, 2 µg/ml chymostatin, 3 µg/ml pepstatin A, 2 µg/ml leupeptin, 1 mM benzamidine-HCl, and 1 mM PMSF) for every gram of tissue. Nuclei, cellular debris and connective tissues were removed by centrifugation at 900 x g for 10 min. For preparation of microsomal membranes, the postnuclear supernatant was subjected to ultracentrifugation at 100,000 x g for 1 h. The pellet fraction was aliquoted and was stored at 80°C until used. After extraction with 140 mM MES buffer (pH 7.0) containing 10% glycerol, 200 mM GlcNAc, 5 mM AMP, and 2% Triton X-100 by sonication, the supernatant was obtained by ultracentrifugation at 100,000 x g for 1 h. Microsomal fractions of various brains and cultured cell lines were obtained in the same manner.
Assay for GlcNAc transferase activity
Assays for GlcNAc transferase activity with respect to the mannosyl peptide, referred to as MGnT activity, were carried out in 1.5 ml Eppendorf tubes in a 50 µl reaction volume containing 140 mM MES buffer (pH 7.0), 200 µM of UDP-[3H]GlcNAc (228,000 dpm/nmol), 10 mM MnCl2, 5 mM AMP, 200 mM GlcNAc, 10% glycerol, 2% Triton X-100, 400 µM synthetic mannosyl peptide and brain microsomal extract. The reaction was initiated by adding the protein extract and continued at 37°C in a shaking water bath for the time given for each experiment. After incubation, the reaction was stopped by boiling for 3 min, and the mixture was filtered through an Ultrafree-MC (0.22 µm pore size, Millipore), and subjected to high-performance liquid chromatography (HPLC) analysis.
The products were fractionated (in 1 ml aliquots) on a Wakopak 5C18-200 column (4.6 x 250 mm, Wako Pure Chemical Ind., Osaka, Japan) at 45°C at a flow rate of 1 ml/min. The column was pre-equilibrated using solvent A. The absorbed proteins were eluted by mixing solvent A with solvent B as follows: 10 min at 0% B; 25 min, linear gradient to 25% B; 5 min, linear gradient to 100% B; 5 min, 100% B; 5 min, linear gradient back to 0% B. The peptide separation was monitored continuously at 214 nm, and the radioactivity of each fraction was measured using a liquid scintillation counter.
The GnT-I activity was measured as described previously (Yoshida et al., 1999). The assay mixture contained 100 mM MES buffer (pH 6.1) 100 mM GlcNAc, 5 mM AMP, 0.2% BSA, 20 mM MnCl2, 1 mM UDP-GlcNAc, 10 µM pyridylaminated oligosaccharide, 0.5% Triton X-100, and yeast extract or brain extract in a total volume of 20 µl. The pyridylaminated acceptor oligosaccharide, Man
1-6(Man
1-3)Man
1-6(Man-
1-3)Man
1-4GlcNAc
1-4GlcNAc-PA (PA017), was obtained from Takara Shuzo (Otsu, Japan). The assay mixture was incubated at 37°C for 1 h. The reaction was stopped by boiling for 3 min, and the mixture was filtered through an Ultrafree-MC (0.22 µm), and subjected to HPLC analysis. A Cosmosil 5C18-AR column (4.6 x 250 mm, Nacalai Tesque, Kyoto, Japan) was equilibrated with a solvent containing 0.15% 1-butanol in 100 mM ammonium acetate. The product was eluted at a rate of 1.2 ml/min at 45°C using the same solvent and was detected by fluorescence of the PA tag: excitation 320 nm and emission 400 nm.
The GnT-II activity was measured as follows. The assay mixture contained 50 mM MES buffer (pH 6.0) 100 mM GlcNAc, 100 mM NaCl, 5 mM AMP, 0.2% BSA, 20 mM MnCl2, 1 mM UDP-GlcNAc, 0.8 mM pyridylaminated oligosaccharide, 1% Triton X-100, and yeast extract or brain extract in a total volume of 10 µl. The acceptor oligosaccharide, Man1-6(GlcNAc
1-2Man
1-3)Man
1-4GlcNAc
1-4GlcNAc-PA, was obtained from human chorionic gonadotropin by hydrazinolysis and derivatized with pyridylamine as described previously (Oguri et al., 1997
). After incubation at 37°C for 1 h, the product was separated as described above, except for the temperature which was at 50°C, using a TSK ODS-80 TM column (4.6 x 150 mm, TOSOH, Tokyo, Japan).
Product characterization
Products were incubated with the following enzyme solutions: (1) jack bean -N-acetylhexosaminidase (0.5 U) in 50 µl of 0.3 M citrate phosphate buffer (pH 5.0) for 18 h at 37°C, and (2) diplococcal
-N-acetylhexosaminidase (50 mU) in 50 µl of 0.3 M citrate phosphate buffer (pH 5.5) for 48 h at 37°C. One drop of toluene was added to the reaction mixtures to inhibit bacterial growth during incubation. After incubation, the enzymes were inactivated by heating the reaction mixture in a boiling water bath for 3 min. The enzyme-digested sample was chromatographed using a Wakopak 5C18-200 column as described above. The passed through fraction was further analyzed using a Shodex SUGAR SP-0810 column in order to determine monosaccharide (Takeuchi et al., 1987
).
-Elimination was performed as follows. The product was dissolved in 500 µl of 0.05 N NaOH and 1 M NaBH4 and incubated for 18 h at 45°C. After adjusting the pH to 5 by adding 4 N acetic acid, the solution was applied to a column containing 1 ml of AG-50W-X8 (H+ form), and the column was then washed with 10 ml of water. The effluent and the washing were combined and evaporated to dryness. After the remaining borate was removed by repeated evaporation with methanol, the residue was subjected to Superdex Peptide HR10/30 column chromatography (Amersham Pharmacia Biotech., Uppsala, Sweden). The radioactive products were eluted with distilled water at a flow rate of 0.4 ml/min at 60°C as described previously (Sato et al., 1999
).
High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed using a Bio-LC system (Dionex Co., Sunnyvale, CA) equipped with a CarboPac PA-1 column (4 x 250 mm). An isocratic elution with 15 mM NaOH was carried out at a flow rate of 1 ml/min at ambient temperature. Reduced disaccharide standards, GlcNAc1-2ManOH, GlcNAc
1-3ManOH, GlcNAc
1-4ManOH, and GlcNAc
1-6ManOH, were obtained from each corresponding disaccharide (Chiba et al., 1997
) after reduction with NaBH4.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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