Glycobiology Advance Access originally published online on May 25, 2005
Glycobiology 2005 15(10):943-951; doi:10.1093/glycob/cwi082
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Characterization of a novel galactose ß1,3-N-acetylglucosaminyltransferase (ß3Gn-T8): the complex formation of ß3Gn-T2 and ß3Gn-T8 enhances enzymatic activity
2 Department of Biochemistry, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; and 3 CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan
1 To whom correspondence should be addressed; e-mail: yamashita{at}sasaki.or.jp
Received on February 23, 2005; revised on May 18, 2005; accepted on May 19, 2005
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Abstract |
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We characterized a novel member of the ß1,3-N-acetylglucosaminyltransferase (ß3Gn-T) gene family, ß3Gn-T8. A recombinant soluble form of ß3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of ß3Gn-T2. The two enzymes had similar substrate specificities and recognized tetraantennary N-glycans and 2,6-branched triantennary glycans in preference to 2,4-branched triantennary glycans, biantennary glycans, and lacto-N-neotetraose (LNnT), indicating their specificity for 2,6-branched structures such as [Galß1
4GlcNAcß12(Galß14GlcNAcß16)Man
1 6Man]. Interestingly, when soluble recombinant ß3Gn-T2 and ß3Gn-T8 were mixed, the Vmax/Km value of the mixture was 9.3- and 160-fold higher than those of individual ß3Gn-T2 and -T8, respectively. Sephacryl S-300 gel filtration of the enzymes revealed that apparent molecular weights of each ß3Gn-T2, ß3Gn-T8, and the mixture were 90–160, 45–65, and 110–210 kDa, respectively, suggesting that ß3Gn-T2 and -T8 can form a complex with enhanced enzymatic activity. This is the first report demonstrating that in vitro mixed glycosyltransferases show enhanced enzymatic activity through the formation of a heterocomplex. These results suggested that ß3Gn-T8 and ß3Gn-T2 are cooperatively involved in the elongation of specific branch structures of multiantennary N-glycans. Key words: N-acetylglucosaminyltransferase / enzyme complex / galactose / Pichia pastoris / tetraantennary N-glycan
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Introduction |
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ß1,3-Linked GlcNAc residues are present in the backbone of various biologically important glycans. For example, GlcNAcß1
3GalNAc1 is the core 3 structure found in many O-linked glycans expressed in the digestive organs (Podolsky, 1985
; Vavasseur et al., 1995). Lacto- and neolacto-series glycolipids contain a GlcNAcß13Galß14Glcß1Cer sequence, which is the backbone for various tumor-specific glycolipid antigens (Hakomori, 1989). Keratan sulfate, a poly-N-acetyllactosamine structure (Galß14GlcNAcß13)n with high 6-O-sulfation, maintains appropriate hydration levels in the cornea and is believed to have a functional role in processes such as cell motility and embryo implantation (Funderburgh, 2000). Lymphocyte homing is initially mediated by L-selectin and its ligand glycans, one of which is a sulfated N-acetyllactosamine on a core 1 glycan (Yeh et al., 2001). Expression of oligo- or poly-N-acetyllactosamine moieties in multiantennary N-glycans has been correlated with baby hamster kidney cell transformation (Yamashita et al., 1984), hepatic carcinogenesis (Yamashita et al., 1989), and granulocyte differentiation (Lee et al., 1990). The variety of ß1,3-linked GlcNAc residues in different types of glycoconjugates and cells at varying stages of differentiation suggests the presence of various ß1,3-N-acetylglucosaminyltransferases (ß3Gn-T) with different substrate specificities and expression profiles in human tissues.
In fact, eight ß3Gn-T genes (iGn-T, ß3Gn-T2, -T3, -T4, -T5, -T6, -T7, and -T8) have been identified thus far, and their activities have been characterized. Several of the enzymes, iGn-T, ß3Gn-T2, -T3, -T4, and -T8, mediate poly-N-acetyllactosamine synthesis (Sasaki et al., 1997; Shiraishi et al., 2001; Ishida et al., 2005), and ß3Gn-T3 is also able to synthesize O-linked core 1 structures (Yeh et al., 2001). ß3Gn-T5, -T6, and -T7 are involved in the synthesis of lactotriose (Togayachi et al., 2001), O-linked core 3 glycans (Iwai et al., 2002), and the keratan sulfate backbone (Kataoka and Huh, 2002; Seko and Yamashita, 2004), respectively. Their substrate specificities can explain which enzymes are responsible for the addition of ß1,3-linked GlcNAc to particular glycoconjugates, but which enzymes contribute to ß1,3-GlcNAc elongation of oligo- or poly-N-acetyllactosamine in multiantennary N-glycans is not yet fully understood.
In this study, we extensively characterized ß3Gn-T8. Although the same gene has been recently designated ß1,3-galactosyltransferase-7 (ß3GALT7) by Huang et al. (2004) without enzymatic characterization, we could not detect any ß3GalT activity in it, and we also redesignated the gene as ß3Gn-T8, like as shown by Ishida et al. (2005). The enzyme acts efficiently on tetraantennary and 2,6-branched triantennary N-glycans, with a specificity similar to that of ß3Gn-T2. Additionally, the in vitro mixing of ß3Gn-T8 and ß3Gn-T2 forms a heterocomplex whose enzymatic activity is greatly enhanced, compared with the individual enzymes.
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Results |
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Characterization of the enzymatic activity of ß3Gn-T8
To analyze the ß3Gn-T activity, we expressed the catalytic domain of the ß3Gn-T8 protein with (His)6 tag sequence by Pichia pastoris KM71 cells. Six transformant colonies were preliminary cultured in a small scale, and the media were assayed for ß3Gn-T activity to select expression-positive clones. Three clones secreted similar levels of ß3Gn-T activity, whereas the other three clones showed no ß3Gn-T activity. In a large-scale preparation as materials and methods, the purified enzyme (T8 fraction) was used as an enzyme source. As for the time dependency, the linearity of the activity was maintained at least for 3 h (data not shown). We similarly prepared a soluble form of ß3Gn-T2, too (T2 fraction). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of T8 and T2 fractions was shown in Figure 1. Both exhibited rather broad bands, and apparent molecular weights of ß3Gn-T8 and -T2 were 50–70 and 80–200 kDa, respectively. After peptide:N-glycanase (PNGase) F digestion, these broad bands were concentrated to 44 kDa (ß3Gn-T8) and 45 kDa (ß3Gn-T2), indicating that large N-glycans were present in yeast-derived ß3Gn-T8 and -T2. The putative molecular weights of the truncated ß3Gn-T2 and -T8 were 45,338 and 42,360, respectively, and the possible N-glycosylation sites of the truncated ß3Gn-T2 and -T8 were five and two, respectively. Large and broad molecular weight of ß3Gn-T2 should be because of heterogeneity of large N-glycans. The GlcNAc-transferase activity of T8 and T2 fractions was 2.0 and 43 nmol/min/mg protein, respectively, using tetraGP as an acceptor.
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The linkage position of GlcNAc was determined using tetraGP as an acceptor. The enzymatic reaction product, [3H]GlcNActetraGP, was purified by paper electrophoresis and paper chromatography. [3H]GlcNAc was ß-linked, because the [3H]GlcNActetraGP bound to a Psathyrella velutina lectin-Sepharose (PVL-Sepharose) column which binds to nonsubstituted ß-GlcNAc residues (Kochibe and Matta, 1989; Endo et al., 1992). Next, the reaction product was ß1,4-galactosylated by bovine milk ß1,4-galactosyltransferase to cap the [3H]GlcNAc residue. When the resulting Galß14[3H]GlcNAcß1tetraGP was digested with Escherichia freundii endo-ß-galactosidase and applied to a Bio-Gel P-4 gel filtration, radioactivity eluted at 4.0 Glc units (two hexose and one HexNAc, data not shown) (Kobata et al., 1987). This indicates that the radioactive product was Galß14[3H]GlcNAcß1Gal, and that Galß14[3H]GlcNAcß1 could be linked to either of the four branching N-acetyllactosamine moieties of the tetraGP.
The [3H]GlcNAc linkage should be at the C-3 position of Gal, based on the substrate specificity of the endo-ß-galactosidase (Fukuda et al., 1984). To further confirm the linkage position, we subjected Galß14[3H]GlcNAcß1tetraGP to periodate oxidation and the Smith degradation. If the [3H]GlcNAc was attached to the C-2, -4, or -6 position of the Gal residue, CHO-CH(O-ß[3H]GlcNAc)-CH2OH, CH2OH-CH(O-ß[3H]GlcNAc)-CHOH-CH2OH, or CH2 (O-ß[3H]GlcNAc)-CHOH-CH2OH, respectively, would be produced by the reactions. In contrast, if the [3H]GlcNAc was attached to the C-3 position of Gal, the theoretical products would be 1-O-([3H]GlcNAcß13Galß14GlcNAcß1)-glycerol, when [3H]GlcNAc is attached to the 6-branch of 1,6-linked Man, or 2-O-([3H]GlcNAcß13Galß1 4GlcNAcß1)-glyceraldehyde, when [3H]GlcNAc is attached to the 2-branch of 1,6-linked Man, or [3H]GlcNAcß1 3Galß14GlcNAcß14(GlcNAcß12)Man13Manß1 4XylNAcol, when the linkage is to the 4-branch of 1, 3-linked Man, or [3H]GlcNAcß13Galß1 4GlcNAcß1 2(GlcNAcß14)Man13Manß14XylNAcol, when the linkage is to the 2-branch of 1,3-linked Man.
The oxidized products were applied to a Bio-Gel P-4 column chromatography (Figure 2). The major 3H-labeled compound (peak II, 61% of the total radioactivity) was eluted at the 6.3 Glc unit position, which corresponds to that of authentic [3H]GlcNAcß13Galß14GlcNAcß1 glycerol. This result indicates that the Smith degradation product is [3H]GlcNAcß13Galß14GlcNAcß1glycerol or glyceraldehyde, and that [3H]GlcNAc introduced by ß3Gn-T8 is attached to the C-3 position of Gal. Accordingly, these data confirm that the enzyme is a ß1,3-GlcNAc-transferase. Peak I (10.8 Glc unit, 8% of the total radioactivity) seemed to be a heptaose, corresponding to authentic [3H]GlcNAc1Gal1GlcNAc2Man2XylNAcol, which should be produced when [3H]GlcNAc is attached to either the 2- or the 4-branch of 1,3-linked Man. The result of the Smith degradation implies that ß3Gn-T8 preferentially acts on the 2,6-branch of 1,6-linked Man of tetraGP.
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To examine whether ß3Gn-T8 has ß1,3-galactosyltransferase (ß3GalT) activity, we used UDP-[3H]Gal as a donor substrate and GlcNAcß13Galß14Glc, agalacto biGP, or GalNAc-O-pNP as acceptor substrates. The reaction mixture was applied to paper electrophoresis and paper chromatography like as ßGn-T assay. No GalT activity was detected for the three acceptor substrates (data not shown), indicating that ß3Gn-T8 does not have ß3-GalT activity.
The optimal pH of the ß3Gn-T8 activity was 7.0–7.5. The addition of ethylenediaminetetraacetic acid (EDTA) (2 mM) instead of 10 mM MnCl2 completely inhibited the enzymatic activity, and the addition of 10 mM CaCl2 or MgCl2 showed no activity, suggesting a requirement of Mn2+ for the enzymatic activity.
The substrate specificity of ß3Gn-T8 is summarized in Table I. ß3Gn-T8 can efficiently act on N-linked glycans (tetraGP and 2,6-branched triGP), and its relative activity is positively correlated with increasing numbers of branch chains. An O-linked-type glycan (Gal-core2-O-pNP) and a glycolipid-type glycan (lacto-N-neotetraose [LNnT]) were also acceptors, but lacto-N-tetraose (LNT) which contains the type 1 chain was a poor substrate. Lacto-N-fucopentaose-III (LNF-III), core 1-pNP, GalNAc-O-pNP, lactosylceramide, galactosylceramide, and L2L2 were also poor substrates (data not shown). These results suggested that ß3Gn-T8 is involved in the elongation of N-acetyllactosamine sequences in various type of glycans, especially in multibranched N-glycans. The transferred position of [3H]GlcNAc to Gal-core2-O-pNP by ß3Gn-T8 was determined by peanut agglutinin-agarose (PNA-agarose) affinity chromatography. PNA binds to the nonreducing terminal of the Galß13(R6)GalNAc structure, but not to the R3Galß13GalNAc (Lotan et al., 1975). The [3H]GlcNAcß13(Gal-core2-O-pNP) completely bound to the PNA-agarose column and was eluted with 0.3 M lactose (data not shown), indicating that the 3H-labeled compound was [3H]GlcNAcß13Galß1 4GlcNAcß16(Galß13)GalNAc1-O-pNP.
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Next, to determine to which branches of biGP [3H]GlcNAc was transferred, we digested [3H]GlcNAcß13biGP with Streptococcus 6646K ß-galactosidase and then was applied to an E4-phytohemagglutinin-agarose (E4-PHA-agarose) column. Galß14GlcNAcß12Man13Manß14GlcNAcß1 is slightly retarded on the lectin column at 2°C, whereas Galß14GlcNAcß12Man16Manß14GlcNAcß1 is more retarded (Kobata and Yamashita, 1989). Because the C-3 substitution at the Gal residue does not affect the elution profile, [3H]GlcNAcß13Galß14GlcNAcß1 2Man16(GlcNAcß12Man13) Manß14GlcNAc should be more retarded on the column than its counterpart having a Man13. As shown in Figure 3, [3H]GlcNAcß1 3biGP was separated into fractions I and II by E4-PHA-agarose chromatography, and fractions I and II should be oligosaccharides containing [3H]GlcNAc on the 13 branch and on the 16 branch, respectively. The ratio of the radioactivities of fractions I and II was 59:41, indicating that ß3Gn-T8 slightly preferred the 13-branching LacNAc to the 16-branching LacNAc as a substrate.
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We were interested whether ß3Gn-T2, which has a strong LacNAc-elongation activity (Shiraishi et al., 2001), can act on multivalent N-linked glycans, similar to the activity of ß3Gn-T8. We examined the substrate specificity of ß3Gn-T2, which was also prepared by KM71 cells as a truncated soluble protein with (His)6 tag sequence. As summarized in Table I, ß3Gn-T2 has a similar substrate specificity to that of ß3Gn-T8, although ß3Gn-T2 had relatively low activities toward 2,4-branched triGP, biGP, monoGP, Gal-core2-pNP, and LNnT.
Both ß3Gn-T8 and ß3Gn-T2 had higher activities for 2,6-branched triGP than for 2,4-branched triGP (Table I). To further confirm this, we measured kinetic values of these enzymes for tetraGP, triGPs, and biGP (Table II). The Vmax/Km values of ß3Gn-T8 for tetraGP and 2,6-branched triGP were higher than those for 2,4-branched triGP and biGP. Similar results were obtained for ß3Gn-T2. These results indicate that a Galß14GlcNAcß1 6Man16Man branch is important for good recognition by the both enzymes.
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Mixing ß3Gn-T8 with ß3Gn-T2 increases enzymatic activity
We found that enzymatic activity in the mixture of ß3Gn-T2 and -T8 was several times higher than the sum of the activities of each enzyme (Figure 4). By the addition of T8 fraction to the reaction mixture of T2 fraction, ß3Gn-T activity was enhanced in comparison with the sum of the individual activities of the two fractions. At 2.2 ng protein of T8 fraction (the relative amount of T8 fraction/T2 fraction = 1.1:1), the activity was six times higher than the sum of the two. Further addition of T8 fraction did not enhance ß3Gn-T activity, suggesting that ß3Gn-T8 may interact with ß3Gn-T2 and that ß3Gn-T8 was saturated at the ratio of T8 fraction/T2 fraction (1.1:1). We thereafter studied character of the enhanced ß3Gn-T activity in the mixture of the two fractions by this ratio. The kinetic values of ß3Gn-T2, -T8, and the mixture of the two enzymes for tetraGP are shown in Table II. The Vmax value of the mixture (470 nmol/min/mg protein) was approximately four times higher than the sum of the each Vmax value (124 nmol/min/mg protein), and the Vmax/Km value of the mixture (1400 nmol/min/mg protein/mM) was approximately nine times higher than that for ß3Gn-T2 (150 nmol/min/mg protein/mM). The substrate specificity of the mixture is summarized in Table I. The specificity of the mixture is similar to that of ß3Gn-T2 and -T8, but is closer in substrate specificity to ß3Gn-T8 than to ß3Gn-T2.
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). Dotted line indicates the predicted sum of the individual activities of 2 ng protein of T2 fraction and indicated amounts of T8 fraction.This activity-enhancing effect was also observed using membrane-bound ß3Gn-T2 and -T8. We prepared crude membrane fractions containing ß3Gn-T2 or -T8 from these expression vector-transfected COS-7 cells and used the fractions as enzyme sources. The Km values for ß3Gn-T2, -T8, and the mixture were almost the same as those for the soluble enzymes (data not shown). The Vmax/Km value for the mixture (480 pmol/min/mg protein/mM) was 4.8- and 15-times higher than those for ß3Gn-T2 (100 pmol/min/mg protein/mM) and ß3Gn-T8 (32 pmol/min/mg protein/mM), respectively. From these results, it was speculated that ß3Gn-T8 binds to ß3Gn-T2 and that the hetero-oligomer has much higher enzymatic activity than either of the individual enzymes.
Gel filtration of soluble ß3Gn-T2, ß3Gn-T8, and the mixture of ß3Gn-T2 and -T8
To determine whether ß3Gn-T2 interacts with ß3Gn-T8, we applied each enzyme alone and the mixture to Sephacryl S-300 gel filtration columns and measured the enzymatic activities of the individual fractions. As shown in Figure 5, the apparent molecular weights of ß3Gn-T2 (peak II) and -T8 (peak III) were estimated to be 90–160 and 45–65 kDa, respectively. In the case of the mixture (peak I), the apparent molecular weight shifted to 110–210 kDa. From the results of SDS–PAGE analysis in Figure 1, the soluble ß3Gn-T2 and -T8 were likely present as a monomer. Similarly, based on the apparent molecular weight, the mixture of ß3Gn-T2 and -T8 was likely to form a heterodimer consisting of one ß3Gn-T2 unit and one ß3Gn-T8 unit.
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), ß3Gn-T8 (
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Discussion |
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We clearly demonstrated in this study that ß3Gn-T8 has similar substrate specificity to that of ß3Gn-T2. ß3Gn-T8 belongs to a ß1,3-GlcNAc-transferase family, based on its ß3Gn-T activity. Huang et al. (2004)
designated the same enzyme as ß3GALT7 without the demonstration of the enzymatic activity. We showed that the enzyme does not have ß3Gal-T activity and designated it as ß3Gn-T8. Recently, Ishida et al. (2005) showed that ß3Gn-T8 has a ß1,3-GlcNAc-transferase activity toward tetraantennary N-glycans. In this study, ß3Gn-T8 and -T2 act most efficiently on 2,6-branching N-acetyllactosamine moieties of tetraGP and 2,6-branched triGP. Soluble ß3Gn-T8 and ß3Gn-T2 formed a heterocomplex when the proteins were mixed in vitro, and the complex exhibits enhanced enzymatic activity in comparison with the individual enzymes.
We used P. pastoris cells for the preparation of soluble forms of ß3Gn-T8 and -T2. This organism is advantageous to use because, unlike Escherichia coli (E. coli), it is able to add N-linked glycans, which are high mannose-type glycans, and, unlike Saccharomyces (S. cerevisiae), the glycans are relatively small. In fact, we were unable to prepare a soluble and enzymatically active form of ß3Gn-T8 and -T2 in E. coli, although soluble, inactive proteins were obtained. Some N-linked glycans of ß3Gn-T8 and -T2 may be essential for proper folding or maintenance of their functional structure. Grinna and Tschopp (1989) reported that P. pastoris synthesizes relatively small high mannose-type N-glycans (average 8–14 mannose residues) in comparison with Saccharomyces cerevisiae (S. cerevisiae). However, our SDS–PAGE data indicated that recombinant ß3Gn-T2 and -T8 have much larger N-glycans than those they reported. The reason for this discrepancy remains unclear, but it is possible that tertiary structure of recombinant proteins may be related to the size of high mannose-type N-glycans.
From the results of the periodate oxidation and substrate specificities, ß3Gn-T8 and -T2 preferentially act on 2,6-branching N-acetyllactosamine moieties. Van den Eijnden et al. (1988) showed that a ß3Gn-T partially purified from Novikoff tumor cell ascites fluid strongly preferred the moieties in tri- and tetraantennary N-glycans. This partially purified enzyme is likely to be ß3Gn-T2, -T8, or the complex of the two enzymes, because the substrate specificity of the enzyme is very similar to that of ß3Gn-T2 and -T8. It has been shown that oligo-N-acetyllactosamine moieties in tri- and tetraantennary N-glycans are present in various glycoproteins including calf thymocyte plasma membrane glycoproteins (Yoshima et al., 1980), plasma 1-acid glycoproteins (Yoshima et al., 1981), human erythropoietins produced in Chinese hamster ovary cells (Sasaki et al., 1987; Takeuchi et al., 1988; Hokke et al., 1995), and lysosomal membrane glycoprotein (LAMP)-1 and LAMP-2 in dimethyl sulfoxide-treated HL-60 cells (Lee et al., 1990). In these cases, N-acetyllactosamine residues elongate preferentially or exclusively at the 2,6-branching. Although it is unknown whether these cells or tissues express ß3Gn-T2 and/or -T8, the glycan structures are consistent with the substrate specificities of ß3Gn-T2 and -T8 found in this study.
The result of Sephacryl S-300 gel filtration indicates that ß3Gn-T2 and -T8 can form a complex. The molecular weights of polypeptide moieties of recombinant soluble ß3Gn-T2 and -T8 are 45,338 and 42,360, respectively, which include an EAEAHHHHHHGSDDDDKYVEF sequence derived from the pPIC9-His expression vector. This is consistent with the results of SDS–PAGE analysis of PNGase F-treated proteins (Figure 1). On the other hand, intact recombinant ß3Gn-T2 and -T8 proteins exhibit rather broad bands on SDS–PAGE with molecular weight 80–200 and 50–70 kDa, respectively (Figure 1). The apparent molecular weight (90–160 kDa) of ß3Gn-T2 alone by gel filtration (Figure 5) should correspond to a monomer form, as that of ß3Gn-T8 alone (45–65 kDa) can be considered to be a monomer. The mixture of the two proteins elutes at 110–210 kDa, suggesting a heterodimer of each ß3Gn-T2 and ß3Gn-T8.
The ß3Gn-T2/T8 complex shows enhanced enzymatic activity in comparison with the individual enzymes, in either the soluble or membrane-bound forms. The substrate specificity of the complex is similar to that of each enzyme. The Vmax value of the complex was consistently much higher than either enzyme alone, although the Km value is comparable with that of ß3Gn-T8. Recently, it has been reported that in vivo co-expression of two individually inactive enzyme proteins resulted in robust enzymatic activity in the cases of core1 ß3GalT and Cosmc (Ju and Cummings, 2002), chondroitin synthase and chondroitin polymerizing factor (Kitagawa et al., 2003), and protein O-mannosyltransferase 1 and 2 (Manya et al., 2004). These enzymatic activities are very low or not detected when mixing individually prepared enzyme fractions in vitro. Ju and Cummings (2002) proposed that Cosmc functions as a molecular chaperone to support active core1 ß3GalT. Our results are quite different from these cases with respect to the enhanced effect obtained by in vitro mixing and the occurrence of the enzymatic activities in individual ß3Gn-T2 and -T8 preparations. Although at present it is unresolved which subunit(s) have enhanced activity, X-ray structural studies should be able to elucidate this issue in the future.
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Materials
UDP-[6-3H]GlcNAc (2.21 TBq/mmol) and UDP-[6-3H]Gal (659 GBq/mmol) were purchased from Perkin Elmer Biosciences (Boston, MA) and Amersham Biosciences (Buckinghamshire, UK), respectively. Fuc
1 3(Galß14) GlcNAcß13Galß14Glc (LNF-III), Galß1 3GalNAc1- O-pNP (core1-O-pNP), and Galß1 3(GlcNAcß16) GalNAc1-O-pNP (core2-O-pNP) were purchased from Funakoshi (Tokyo, Japan). Bovine milk ß1,4-galactosyltransferase, GalNAc1-O-pNP, and UDP-GlcNAc were purchased from Sigma (St. Louis, MO). Galß1 3GlcNAcß13Galß14Glc (LNT) and Galß1 4GlcNAcß13Galß14Glc (LNnT) were prepared from human milk (Kobata, 1972). Ricinus communis agglutinin-I-agarose (RCA-I-agarose) (4 mg/mL gel) was obtained from Hohnen Oil (Tokyo, Japan). PVL was prepared according to the method described by Kochibe and Matta (1989), and the lectin was conjugated to CNBr-activated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions. PNA-agarose (4.5 mg/mL gel) was purchased from E-Y Laboratories (San Mateo, CA). Galß14GlcNAcß12Man13(6)Manß14GlcNAc (monoGP), Galß14GlcNAcß12Man13(Galß1 4GlcNAcß12Man16)Manß14GlcNAc (biGP), a mixture of Galß14GlcNAcß12 (Galß14GlcNAcß14) Man13(Galß14GlcNAcß12Man16)Manß1 4GlcNAc (2,4-branched triGP) and Galß14GlcNAcß1 2Man13[Galß14GlcNAcß12(Galß14GlcNAcß16) Man16]Manß14GlcNAc (2,6-branched triGP), and Galß14GlcNAcß12(Galß14GlcNAcß14) Man1 3 [Galß14GlcNAcß12 (Galß14GlcNAcß1 6)Man1 6]Manß14GlcNAc (tetraGP) were obtained from the urine of GM1 gangliosidosis patients (Yamashita et al., 1981). Galß14(SO3–6)GlcNAcß13Galß1 4(SO3–6)GlcNAc (L2L2) was the kind gift of Seikagaku (Tokyo, Japan). Streptococcus 6646K ß-galactosidase, E. freundii endo-ß-galactosidase, and E4-PHA-agarose (3.6 mg/mL gel) were obtained from Seikagaku.
cDNA cloning of ß3Gn-T8
Based on the amino acid sequence of human ß3Gn-T2 (Shiraishi et al., 2001), we found one sequence [GenBank, AY277592
[GenBank]
, submitted by Huang et al. (2004)] in the NCBI database (NIH, Bethesda, MD) with a high degree of similarity. The cDNA encoding the full open reading frame was amplified by polymerase chain reaction (PCR) from QUICK-CloneTM c DNA for human colon adenocarcinoma (Clontech, Palo Alto, CA). The oligonucleotide primers used were 5'-tttaagcttATGCGCTGCCCCAAGTG-3' (forward primer) and 5'-ttttctagaTCAGCACTGGAGCCTTG-3' (reverse primer). The sequences in lowercase letters contain appropriate restriction sites. Amplified cDNA was digested with HindIII and XbaI and cloned into pcDNA3 (Invitrogen Life Technologies, Carlsbad, CA). The constructed plasmid was named pcDNA3-ß3Gn-T8 and sequenced using a PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Similarly, the cDNA encoding ß3Gn-T2 was amplified by PCR using the oligonucleotide primers 5'-tttaagcttGAGAAATGAGTGTTGGA-3' (forward primer) and 5'-ttttctagaACACAACATGGGAAC-3' (reverse primer) and was cloned into pcDNA3 between the same sites as ß3Gn-T8 (pcDNA3-ß3Gn-T2).
Expression of ß3Gn-T8 and -T2 in COS-7 cells was performed, as described previously (Seko et al., 2001). The crude membrane fraction was used for examining the enzymatic activity.
Expression of soluble forms of ß3Gn-T2 and ß3Gn-T8 in P. pastoris
The cDNA fragments of truncated forms of ß3Gn-T2 and ß3Gn-T8, lacking cytoplasmic and transmembrane domains, were amplified by PCR using with pcDNA3-ß3Gn-T2 and -T8, respectively. The oligonucleotide primers used were 5'-tttgaattcTCCAAAAGCAGTAGCC-3' (forward primer for ß3Gn-T2), 5'-tttgcggccgcTAATGTGAGAACACAAC-3' (reverse primer for ß3Gn-T2), 5'-tttgaattcAGCAAGGCCTACCC-3' (forward primer for ß3Gn-T8), and 5'-tttgaattcAGCACTGGAGCCTTG-3' (reverse primer for ß3Gn-T8). An expression vector, pPIC9-His, was produced from pPIC9 (Invitrogen Life Technologies) by the insertion of 5'-CATCACCATCACCATCACGGATCCGATGACGATGACAAA-3' sequence at the upstream of SnaB1 site. The cDNAs were cloned into pPIC9-His between EcoRI and NotI for ß3Gn-T2 and at the EcoRI site for ß3Gn-T8. The resulting plasmids were sequenced with a Prism 310 Genetic Analyzer.
Production of the recombinant proteins in culture media of P. pastoris was performed using a Pichia Expression Kit, Version M (Invitrogen Life Technologies). Briefly, the plasmids were linearized with SalI and used for the transformation of Pichia KM71 cells. The transformants were inoculated in 300 mL of buffered glycerol-complex medium and cultured at 30°C at 230 rpm for 3 days. After centrifugation at 1500 g for 5 min, cell pellets were resuspended in 100 mL of buffered methanol-complex medium containing 1% casamino acids and cultured at 28°C at 230 rpm for 2 days. Methanol (0.5 mL each) was added every day to maintain induction. The cultures were centrifuged at 5000 rpm for 10 min at 4°C. The supernatants were collected, and final concentrations of 1 mM phenylmethanesulfonyl fluoride, 1 µg/mL pepstatin, 1 µg/mL leupeptin, and 0.02% bovine serum albumin (BSA) were added. The solutions were dialyzed against 10 mM sodium phosphate and 0.15 M NaCl (pH 8.0) and then adjusted to final concentration of 5 mM imidazole (pH 8.0). The solutions were applied to nickel-nitrilotriacetic acid (Ni-NTA) agarose (0.7 x 1.3 cm; equilibrated with 20 mM sodium phosphate, 0.3 M NaCl, and 10 mM imidazole [pH 8.0] [buffer A]) (QIAGEN GmbH, Hilden, Germany). The columns were washed with buffer A, and the proteins were eluted with buffer A containing 0.25 M imidazole. The eluates were concentrated with Ultrafree-PFL (NMWL: 10,000) (Millipore, Yonezawa, Japan) and washed repeatedly with 10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES)-NaOH, 0.15 M NaCl, and 10% glycerol (pH 7.2). The concentrates (0.3 mL) for ß3Gn-T2 and -T8 were named T2 fraction and T8 fraction, respectively, and were used for measurement of ß3Gn-T activities. Purified enzymes were analyzed by SDS–PAGE followed by staining with SYPRO Orange (Molecular Probes, Eugene, OR) and detected with FLA-2000 (Fuji Photo Film, Tokyo, Japan). PNGase F (Takara Shuzo, Kyoto, Japan) digestion was performed with denatured condition according to the manufacturer’s instructions, and the digests were analyzed by SDS–PAGE. Protein concentrations were estimated by SDS–PAGE and SYPRO Orange staining using BSA as a standard.
Assay of ß3Gn-T activity
As for T2 and T8 fractions, a reaction mixture (20 µL) consisted of 50 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 0.1% (w/v) Triton X-100, 0.5 mM acceptor substrate, 2.5 µM UDP-[3H]GlcNAc (6.7 x 106 dpm), 50 µg/mL protamine chloride, 0.5 mM spermine, and appropriately diluted enzyme fractions. When assaying the activities of mixed enzymes, enzyme mixtures without substrates were preincubated on ice for 20 min. As for COS-7-derived membrane fractions, a reaction mixture (20 µL) consisted of 50 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 0.5% (w/v) Triton X-100, 0.5 mM acceptor substrate, 2.5 µM UDP-[3H]GlcNAc (6.7 x 106 dpm), 30 µM UDP-GlcNAc, 0.1 M GlcNAc, 1 mM adenosine S'-phosphate, and appropriately diluted enzyme fractions. The reaction mixture was incubated at 37°C for 3 h. The 3H-labeled products were purified by paper electrophoresis (pyridine : acetic acid : water, 3:1:387, pH 5.4) and then by paper chromatography (pyridine : ethyl acetate : acetic acid : water, 5:5:1:3). After drying, the paper was monitored for radioactivity with a radiochromatogram scanner, and the 3H-labeled products were extracted with water and counted. When using glycolipids as acceptors, the reaction mixture was applied to Sep-Pak C18 cartridges (Waters, Milford, MA), and the 3H-labeled products were eluted with methanol.
Characterization of the 3H-labeled product
The enzymatic reaction of ß3Gn-T8 was performed using the tetraGP as an acceptor substrate. The [3H]GlcNActetraGP was purified by paper electrophoresis and paper chromatography as above and then was galactosylated by bovine milk ß1,4-galactosyltransferase (Sigma) in a 30 µL-reaction mixture containing 50 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 15 mU ß1,4-galactosyltransferase, [3H]GlcNActetraGP, and 200 µM UDP-Gal at 37°C for 16 h. The galactose-capped product was purified by paper electrophoresis and paper chromatography and was subjected to periodate oxidation and the Smith degradation (Spiro, 1966; Seko et al., 2001). Briefly, the radioactive products were dissolved in 30 µL of 75 mM sodium acetate (pH 5.3) and 75 mM sodium periodate and incubated at 4°C for 48 h in the dark. Three microliters of 20% ethyleneglycol was added and incubated at 25°C for 3 h. The mixture was added to 300 µL of 0.1 M sodium borate (pH 9.0), 0.1 M sodium borohydride and incubated at 25°C overnight. After acidification with 1 M acetic acid, the mixture was applied to a Bio-Rad AG50W-X8 (H+ form) column (Bio Rad, Hercules, CA), and the flow through fraction was evaporated repeatedly with methanol. The residues were hydrolyzed in 100 µL of 0.05 N H2SO4 at 80°C for 1 h. After neutralizing with 1 N NaOH, the mixture was desalted with AG50W-X8 (H+ form) and AG1-X8 (OH– form) and applied to a Bio-gel P-4 (<45 µm) gel filtration column (1.5 x 50 cm), equilibrated and eluted with distilled water at 55°C.
Separation of 2,6-branched triGP from 2,4-branched triGP
A triGP mixture (500 µg) was applied to an L4-phytohemagglutinin-agarose (L4-PHA-agarose) column (4.1 mg/mL gel; 0.9 x 7.9 cm) (Seikagaku), which was equilibrated and eluted with 10 mM Tris–HCl (pH 8.0), 0.15 M NaCl, 1 mM CaCl2, and 1 mM MgCl2 (Buffer A). L4-PHA has a weak affinity for 2,6-branched glycans such as [Galß14GlcNAcß12(Galß14GlcNAcß16)Man16Manß14GlcNAc] (Cummings and Kornfeld, 1982; Kobata and Yamashita, 1993). The glycans were applied to the L4-PHA column at 4°C, and after washing with 10 mL of Buffer A, the column was stored at room temperature and washed with 20 mL of Buffer A. The retarded fraction was eluted at room temperature and contained the 2,6-branched triGP. The flow through fraction, eluted at 4°C, was applied to a concanavalin A-Sepharose (Con A-Sepharose) column (10 mg/mL of gel; 0.9 x 7.9 cm) (Amersham Biosciences), which was equilibrated and eluted with Buffer A, to remove biantennary glycans possessing three N-acetyllactosamine units (Yamashita et al., 1981). The flow through fraction contained the 2,4-branched triGP. The purified triGPs were desalted with Sephadex G-25 gel filtration columns (1.4 x 68 cm), which were equilibrated and eluted with 5% ethanol, and used for ß3Gn-T substrates in enzymatic activity assays.
Preparation of Gal-core2-O-pNP
A reaction mixture (7 mL) containing 2 mM core2-O-pNP, 50 mM HEPES-NaOH (pH 7.2), 10 mM MnCl2, 60 µg/mL bovine milk ß1,4-galactosyltransferase, and 2.5 mM UDP-Gal was incubated at 37°C for 16 h. After heating at 100°C for 5 min, the reaction mixture was applied to an RCA-I-agarose column by divided eight times (2.3 x 6.5 cm; equilibrated with 10 mM Tris–HCl [pH 8.0], 0.15 M NaCl, and eluted with the same buffer containing 10 mM lactose). The bound fractions were applied to a Sep-Pak C18 column to remove salts and lactose. Finally, 7.3 µmol of Gal-core2-O-pNP was obtained.
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Abbreviations |
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ß3Gn-T, ß1,3-N-acetylglucosaminyltransferase; ß3Gal-T, ß1,3-galactosyltransferase; E4-PHA, E4-phytohemagglutinin; HEPES-NaOH, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid-NaOH; L4-PHA, L4-phytohemagglutinin; LNnT, lacto-N-neotetraose; PCR, polymerase chain reaction; P. pastoris, Pichia pastoris; PNA, peanut agglutinin; PNGase, peptide:N-glycanase; pNP, p-nitrophenyl; PVL, Psathyrella velutina lectin; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis
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