Glycobiology, 2001, Vol. 11, No. 1 37-45
© 2001 Oxford University Press
A new 
-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans
Department of Glycobiology, Tokyo Metropolitan Institute of Gerontology, 35–2 Sakaecho, Itabashi-ku, Tokyo 173–0015, Japan, 2KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1–13–5 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236–0004, Japan, 3Research Department, The Noguchi Institute, 1–8–1 Kaga, Itabashi-ku, Tokyo 173–0003, Japan, and 4Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4–6–1 Shirokanedai, Minato-ku, Tokyo 108–8639, 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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Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc
1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose -1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to -linked mannose residues, and the formed linkage was confirmed to be 1-2 on the basis of diplococcal -N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: 
-3-D-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: -6-D-mannoside -1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.
Key words: GlcNAc transferase/GlcNAc1-2/GnT-I/GnT-II/O-mannosyl glycan
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Proteins produced by eukaryotic cells are frequently post-translationally modified by adding carbohydrates. Carbohydrate moieties of these glycoproteins play important roles not only in modulating properties, such as protein stability and conformation, but are also the key elements in various molecular recognition processes, such as bacterial and viral infection, cell adhesion in the presence of inflammation and metastasis, cell differentiation and development, and many other events characterized by intercellular communication (Kobata, 1992
; Varki, 1993). Understanding the carbohydrate recognition process is important, but the precise mechanisms underlying many carbohydrate-mediated recognition processes are not well understood.
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 GlcA1-6Man 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 (Sia2-3Gal1-4GlcNAc1-2Man, Gal1-4GlcNAc1-2Man, Gal1-4(Fuc1-3)GlcNAc1-2Man, HSO3-3GlcA1-3Gal1-4GlcNAc1-2Man) 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 GlcNAc1-2Man 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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Detection of transferase activity of GlcNAc from UDP-GlcNAc against a synthetic mannosyl peptide
Initially, we attempted to develop an assay for GlcNAc transferase activity with respect to the mannosyl residue of a synthetic octapeptide (Ala-Thr-Pro-Thr(Man)-Pro-Val-Thr-Ala), which corresponds to 316–323 amino acids of
-dystroglycan (Ibraghimov-Beskrovnaya et al., 1992). Since the exact mannosyl glycan attachment sites are not identified yet, the position of mannosyl-threonine was selected randomly. Using this synthetic peptide, we observed a significant incorporation of radioactivity into the mannosyl peptide in the neonatal rat brain extract. However, when we used another synthetic octapeptide which did not contain the mannosyl residue, (Ala-Thr-Pro-Thr-Pro-Val-Thr-Ala), radioactivity incorporation was also observed (data not shown). These results indicate that the majority of radioactivity incorporated was independent of the presence or absence of the mannosyl residue. At this stage, we assumed that incorporation of radioactivity was probably due to the activity of UDP-N-acetylglucosamine: peptide N-acetylglucosaminyltransferase (O-GlcNAc transferase), which transfers GlcNAc from UDP-GlcNAc to the OH residue on Ser or Thr (Hart, 1997). Therefore, we synthesized a different mannosyl peptide by replacing the potential O-GlcNAc glycosylation sites with alanine (Ac-Ala-Ala-Pro-Thr(Man)-Pro-Val-Ala-Ala-Pro-NH2). Since we observed high degradation of the synthetic peptide during incubation, we blocked both N- and C-termini as described in Materials and methods to prevent degradation of the peptide.
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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Most of the glycosyltransferases studied to date require divalent cations as activators (Schachter, 1994
). The divalent ion requirement for MGnT activation was also studied, and the results are shown in Figure 3. The results clearly showed that the enzyme apparently did require a divalent metal ion for its activity, because it was inactive when assayed in the presence of EDTA. Among the metal ions examined, manganese ion showed the highest ability to activate. When assayed in the absence of divalent metal ions, slight activity could be observed probably due to contaminant ions during the preparation. Calcium and magnesium ions served as an activator of the enzyme, but to a lesser extent than manganese ion (Figure 3).
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Identification of product by MGnT
In order to characterize the product, the radioactive product was subjected to conventional
-elimination, which released O-linked sugar chains from the polypeptide backbone. Tritium-labeled O-linked oligosaccharides released by alkaline -elimination from the product were separated by Superdex peptide column chromatography. A single peak, which was eluted at the same retention time as that of authentic Gal1-4GlcNAcOT, was observed as shown in Figure 4. These results indicate that the GlcNAc residue was attached on the mannosyl peptide via a mannosyl residue.
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4GlcNAcOT.In order to characterize the product further, the radioactive mannosyl peptide was directly digested by two
-N-acetylhexosaminidases. As shown in Figure 5A, most of the radioactivity was released from the glycopeptide by treatment with the jack bean enzyme. The result indicates that the GlcNAc residue is linked to the Man residue by a -configuration not an -configuration. However, the exact linkage between the GlcNAc and Man residues could not be determined at this stage because -N-acetylhexosaminidase derived from jack bean can cleave all GlcNAc -linkages (Li and Li, 1972). When the product was incubated with diplococcal -N-acetylhexosaminidase, which cleaves the GlcNAc1-2Man linkage but not the GlcNAc1-4Man or GlcNAc1-6Man linkage (Yamashita et al., 1981), it could also release most of the radioactivity from the glycopeptide (Figure 5B). At this stage, at least we can conclude that the GlcNAc-Man linkage formed is neither GlcNAc1-4Man nor GlcNAc1-6Man. However, since it remains unclear whether or not diplococcal -N-acetylhexosaminidase can cleave the GlcNAc1-3Man linkage, we cannot conclude that the linkage formed here is GlcNAc1-2Man, GlcNAc1-3Man, or both.
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Next, we determined whether or not the released radioactive HexNAc is GlcNAc or other monosaccharides. Both pass through fractions in Figure 5 were further analyzed by HPLC. All the radioactive components were eluted at the same retention time as that of the authentic N-acetylglucosaminitol (GlcNAcOH) standard as shown in Figure 6. Based on these results, we considered that the GlcNAc residue is linked to the 2- and/or 3-position of Man residue by a
-configuration.
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To determine the GlcNAc-Man linkage,
1-2, 1-3, or both, the above -eliminated product (disaccharide alditol) was analyzed by HPAEC-PAD. For identification of the GlcNAc-Man linkage of the -eliminated product (Figure 4), we separated four possible linkage isomers of GlcNAc-ManOH by HPAEC-PAD, and found out that isocratic elution with 15 mM NaOH is effective to separate a mixture of four standards into three peaks; GlcNAc1-6ManOH, a mixture of GlcNAc1-4ManOH and GlcNAc1-3ManOH, and GlcNAc1-2ManOH (Figure 7). When the -eliminated radioactive product mixed with the standard alditol was subjected to HPAEC-PAD, it was eluted at the same position of the authentic GlcNAc1-2ManOH (Figure 7). Taken together, we conclude that the GlcNAc residue is linked to the 2-position of the Man residue on the peptide.
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MGnT is different from GnT-I and GnT-II
Two GlcNAc transferases that catalyze the formation of the GlcNAc
1-2Man linkage, GnT-I and GnT-II, have been purified and cloned (Schachter, 1994). Both GnT-I and GnT-II are essential to the biosynthesis of complex N-glycans (Schachter, 1994). In the present study, we detected the activity associated with formation of the GlcNAc1-2Man linkage. Therefore, it is of great importance to determine whether or not the GlcNAc1-2Man formation observed here is by GnT-I and/or GnT-II. For such a purpose, we used yeast cell lines transformed by the GnT-I or GnT-II gene (Yoshida et al., 1999; unpublished observations), and the MGnT activity in both transfectants was examined. Neither GnT-I nor GnT-II was found to function as a relevant GlcNAc transferase to the mannosyl peptide in our assay systems (Table I). The results clearly indicate that GlcNAc1-2Man formation found in the rat brain is not due to the action of conventional GnT-I or GnT-II.
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Distribution of MGnT activity
To determine the distribution of this novel MGnT, we examined its activity in brains of several species and two cell lines. As shown in Table II, all the brains and cell lines examined showed the transferase activity, more or less. It is noteworthy that all products are concluded to have the GlcNAc
1-2Man linkage because the same analytical data of each product were obtained (data not shown). It suggests that this enzyme may be distributed ubiquitously in mammals although the details remain to be determined.
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Discussion |
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In mammals, a few glycoproteins were modified by O-linked Man: rat brain proteoglycans,
-dystroglycan, and mouse J1/tenascin (Endo, 1999). A series of mammalian-type O-mannosyl glycans, with heterogeneous Man-branching (2- or 3-substituted and 2,6-disubstituted mannoses) and peripheral structures (Sia2-3Gal1-4GlcNAc1-2Man, Gal1-4GlcNAc1-2Man, Gal1-4(Fuc1-3)GlcNAc1-2Man, HSO3-3GlcA1-3Gal-1-4GlcNAc1-2Man) have been found in previous studies (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). Among them, -dystroglycans from different species and different tissues, for example, bovine peripheral nerve, rabbit skeletal muscle, and sheep brain (Chiba et al., 1997; Sasaki et al., 1998; Smalheiser et al., 1998), all contain O-mannosyl glycans, suggesting its relevance to the basic role of -dystroglycan.
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 GlcNAc1-2Man of N-glycans is essential to cell–cell interactions and morphogenesis of multicellular organisms. This step is necessary to generate diverse structures. Thus, the investigation of GlcNAc1-2Man 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 GlcNAc1-3Man 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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Materials
UDP-[3H]GlcNAc (38.5 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Unlabeled UDP-GlcNAc, adenosine monophosphate (AMP) and Triton X-100 were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan).
-N-Acetylhexosaminidase, which was purified from jack bean meal, was purchased from Seikagaku Corp., (Tokyo, Japan). Another -N-acetylhexosaminidase, which was purified from Diplococcus pneumoniae, was purchased from Boehringer Mannheim (Mannheim, Germany).
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 W303–1A (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, Man1-6(Man1-3)Man1-6(Man-1-3)Man1-4GlcNAc1-4GlcNAc-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(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4GlcNAc-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, GlcNAc1-3ManOH, GlcNAc1-4ManOH, and GlcNAc1-6ManOH, were obtained from each corresponding disaccharide (Chiba et al., 1997) after reduction with NaBH4.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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We thank Dr. Satoshi Yoshida for the GnT-I-transformed yeast strain and expression vector of GnT-II, Ms. Nagako Kawashima for technical assistance, and Dr. Akira Kobata for encouraging this work. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (A) [10178102 (TE); 11212236 (TI)] from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the New Energy and Industrial Technology Development Organization (NEDO) and Sumitomo Marine Welfare Foundation for Welfare of Aged People.
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Abbreviations |
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Fmoc, 9-fluorenyloxymethylcarbonyl; GnT-I, UDP-N-acetylglucosamine:
-3-D-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GnT-II, UDP-N-acetylglucosamine: -6-D-mannoside -1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143); Gal, galactose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; HPLC, high-performance liquid chromatography; Man, mannose; MGnT, UDP-N-acetylglucosamine: O-linked mannose -1,2-N-acetylglucosaminyltransferase; PA, pyridylamine; TFA, trifluoroacetic acid; subscript OT and OH are used to indicate NaB3H4- and NaBH4-reduced mono- or disaccharides, respectively.
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1 To whom correspondence should be addressed
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