Purification and cDNA cloning of UDP-GlcNAc:GlcNAcß1-3Galß1-4Glc(NAc)-R [GlcNAc to Gal]ß1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

Hiroaki Korekane3, Tomohiko Taguchi3,4, Yoshihiro Sakamoto3, Koichi Honke3, Naoshi Dohmae5, Heidi Salminen6, Suvi Toivonen6, Jari Helin6, Koji Takio1,5, Ossi Renkonen6 and Naoyuki Taniguchi23

3 Department of Biochemistry, Osaka University Medical School/graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA
5 Biomolecular Characterization Division, Riken, Wako, Saitama 351-0198, Japan
6 The Institute of Biotechnology, University of Helsinki, FIN-00014 Helsinki, Finland

Received on November 28, 2002; revised on December 25, 2002; accepted on January 5, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A rat intestinal ß1,6N-acetylglucosaminyltransferase (ß1-6GnT) responsible for the formation of the ß1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcß1-3Galß1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcß1-3(GlcNAcß1-6)Galß1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcß1-3'LacNAc into GlcNAcß1-3'(GlcNAcß1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcß1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galß1-3GalNAc{alpha}1-O-paranitrophenyl (pNP) and GlcNAcß1-3GalNAc{alpha}1-O-pNP, into Galß1-3(GlcNAcß1-6) GalNAc{alpha}1-O-pNP (C2GnT activity) and GlcNAcß1-3(GlcNAcß1-6)GalNAc{alpha}1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 ß1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I–branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.

Key words: ß1,6N-acetylglucosaminyltransferase / I antigen / mucin core 2 structure / mucin core 4 structure / poly-N-acetyllactosamine


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Linear and branched poly-N-acetyllactosamine chains, which consist of repeating N-acetyllactosamine units, are present in glycoproteins, glycolipids, and proteoglycans. In the case of linear poly-N-acetyllactosamine chains, the N-acetyllactosamine units are linked via ß1,3 linkages. In branched glycans, some of the 3-O-substituted Gal residues in the primary chains are also substituted at position 6 by additional N-acetyllactosamine units. Linear and branched poly-N-acetyllactosamino-glycan backbones correspond to the blood group i and I antigens, respectively (Kemler et al., 1977Go; Niemann et al., 1978Go; Watanabe et al., 1979Go), the expression of which is known to be regulated in the development of fetal to adult erythrocytes (Fukuda et al., 1979Go, 1984aGo,bGo), in the course of murine embryonic development (Kapadia et al., 1981Go), and in embryonal carcinoma cells (Muramatsu et al., 1978Go).

It is known that two types of ß1,6N-acetylglucosaminyltransferase (ß1-6GnT) activities act on linear poly-N-acetyllactosamine chains, leading to the generation of the blood group I antigen structure. The first type of enzyme activity acts at the (growing) ends of poly-N-acetyllactosamine chains, converting GlcNAcß1-3Galß1-4GlcNAcß1- R to GlcNAcß1-3(GlcNAcß1-6)Galß1-4GlcNAcß1-R (Brockhausen et al., 1986Go; Gu et al., 1992Go; Helin et al., 1997Go; Koenderman et al., 1987Go; Piller et al., 1984Go; Ropp et al., 1991Go; Vanderplasschen et al., 2000Go; Yeh et al., 1999Go). We refer to this enzyme activity as distally acting I-branching ß1,6N-acetylglucoaminyltransferase (dIGnT) activity to emphasize the distal site of its action (Leppänen et al., 1998Go). The second type of enzyme activity acts at the midchain positions of the (completed or growing) poly-N-acetyllactosamine chains, converting Galß1-4GlcNAcß1-3Galß1-4GlcNAcß1-R to Galß1-4GlcNAcß1-3(GlcNAcß1-6)Galß1-4GlcNAcß1-R (Bierhuizen et al., 1993Go; Chen et al., 2000Go; Gu et al., 1992Go; Leppänen et al., 1991Go, 1997bGo; Mattila et al., 1998Go; Sakamoto et al., 1998Go; Ujita et al., 1999Go). Accordingly, we refer to this enzyme activity as centrally acting I-branching ß1,6N-acetylglucoaminyltransferase (cIGnT) activity to emphasize the central site of its action (Leppänen et al., 1998Go). None of the cIGnTs purified or cloned to date have been found to have substantial dIGnT activity (Chen et al., 2000Go; Mattila et al., 1998Go; Sakamoto et al., 1998Go; Ujita et al., 1999Go).

Thus far, several reports (Ropp et al., 1991Go; Schwientek et al., 1999Go; Vanderplasschen et al., 2000Go; Yeh et al., 1999Go) have appeared concerning the nature of proteins that have dIGnT activity. The purification of dIGnT was carried out from bovine trachea, but the scarcity of the purified material hampered its cDNA cloning (Ropp et al., 1991Go). Apart from that study, two groups recently reported on the cDNA cloning of a protein with dIGnT activity (Schwientek et al., 1999Go; Yeh et al., 1999Go). They found that the human homolog of leukocyte-type core 2 ß1-6GnT (C2GnT-L; Bierhuizen and Fukuda, 1992Go) possesses dIGnT activity, as well as core 4 ß1-6GnT activity (Schwientek et al., 1999Go; Yeh et al., 1999Go). The original C2GnT-L was shown to have neither dIGnT nor core 4 ß1-6GnT activity (C4GnT; Bierhuizen and Fukuda, 1992Go; Yeh et al., 1999Go). This newly identified homolog was named mucin-type core 2 ß1-6GnT (C2GnT-M) because it is mainly expressed in mucin producing tissues at the mRNA level (Yeh et al., 1999Go). An ortholog of C2GnT-M was identified in bovine herpes virus type 4, which was also shown to have dIGnT and C4GnT activities (Vanderplasschen et al., 2000Go).

In this article we describe the purification and cloning of dIGnT protein with the aim of revealing what dIGnT protein actually functions in tissues. Rat small intestine was chosen, instead of bovine trachea (Ropp et al., 1991Go) in terms of the stability of this enzyme, which is a crucial factor in protein purification. The enzyme was purified to apparent homogeneity by successive column chromatographic procedures using Q-Sepharose FF, Ni2+-chelating Sepharose FF, Zn2+-chelating Sepharose FF, UDP-hexanolamine-agarose, and lacto-N-triose II-aminocellulofine. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The predicted protein sequence showed a significant homology with those of human and viral C2GnT-Ms, indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Purification of dIGnT
The activity of dIGnT was assayed using lacto-N-triose II-2-aminopyridine (PA) as an acceptor substrate, basically according to the method of Gu et al. (1992)Go. Bovine serum albumin (BSA) (2 mg/ml) was found to be effective in preserving enzyme activity at 37°C, especially for the highly purified enzyme fractions (steps 6 and 7). The activity of these purified fractions was 30-fold higher than that under the assay conditions in the absence of BSA. As a result, BSA was routinely included in the standard assay mixture.

Rat small intestine was chosen as an enzyme source for the following reasons. First, a survey of various rat and hog tissues revealed that rat small intestine and hog gastric mucosa contained high levels of dIGnT activity per tissue protein. Second, dIGnT activity from hog gastric mucosa, after solubilization with Triton X-100, is only associated with an extremely high molecular weight fraction, and none of the column chromatographies were useful for its purification.

Like other GnTs, the rat small intestinal dIGnT was concentrated in the microsome fraction. At least 80% of the activity was associated with the microsome fraction. The dIGnT activity from the microsome fraction was solubilized by Triton X-100 more effectively at pH 7.4 than at pH 9.0 (Gu et al., 1992Go). The majority of proteins were separated from the dIGnT activity by Q-Sepharose FF, Ni2+-chelating Sepharose FF, and Zn2+-chelating Sepharose FF column chromatographies (Figure 1A, B, and C).



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Fig. 1. Elution patterns for the chromatographic steps used in the purification of dIGnT. (A) Q-Sepharose FF column chromatography (step 3); (B) Ni2+-chelating Sepharose FF column chromatography (step 4); (C) Zn2+-chelating Sepharose FF column chromatography (step 5). (D) UDP-hexanolamine-agarose column chromatography (step 6); (E) Lacto-N-triose II-aminocellulofine column chromatography (step 7); (F) SDS–PAGE (8.5% gel) of the fractions eluted from the lacto-N-triose II-aminocellulofine column under nonreducing conditions, followed by silver staining. Fractions indicated by bars were pooled.

 
After the Ni2+-chelating Sepharose FF column chromatography (step 4), dIGnT activity was more stable than in steps 2 and 3. Almost all of the cIGnT activity was separated from the dIGnT activity by the Zn2+-chelating Sepharose FF column chromatography. The use of an affinity column (step 6, Figure 1D) packed with an analog of the common donor substrate for GnTs (UDP-hexanolamine) as a ligand was the most effective. This affinity column has been proven to be very effective for the purification of GnTs and was first used for the purification of GnT I by Oppenheimer and Hill (1981)Go. dIGnT activity was completely bound to the UDP-hexanolamine-agarose column and was sharply eluted by UDP as a competitive ligand. Different from cIGnT and GnT VI (Sakamoto et al., 1998Go; Taguchi et al., 2000Go), the addition of dithiothreitol (0.5 mM) was not effective in preserving dIGnT activity in this affinity column chromatography. The eluted enzyme fraction (after step 6) still contained several proteins of various molecular weights, as evidenced by silver staining. Neither cation exchange-, dye affinity-, gel filtration-, lectin-, nor UDP-GlcNAc-5-propylamine column chromatography was effective in the further purification of the enzyme fraction after step 6.

The final purification was achieved by a second affinity column chromatography (step 7, Figure 1E), using an acceptor substrate (lacto-N-triose II) as an affinity ligand. More than half of the dIGnT activity was bound to the lacto-N-triose II-aminocellulofine column and was eluted with the buffer containing NaCl. When the flow-through fraction was reapplied to the regenerated affinity column, no further binding was observed (data not shown), indicating that the column was not overloaded. The enzyme fractions eluted in step 7 were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing conditions, followed by silver staining (Figure 1F). The protein band with a molecular weight of 66,000 was correlated exactly with the enzyme activity, indicating that this protein was dIGnT. The enzyme fraction eluted with 0.1 M NaCl was used for the enzyme characterization described below. No GnT III, IV, V, cIGnT, or iGnT (ß1-3GnT) activity was detected in this fraction. The protein concentration in this fraction (Step 7) could not be determined due to its low concentration, below the detection limit of the protein assay kit. Table I summarizes the purification of dIGnT, which was purified 45,000-fold in 0.6% yield by steps 1 through 6.


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Table I. Purification of dIGnT from rat small intestine

 
pH optimum of the purified rat dIGnT
The purified rat dIGnT was most active between pH 7.0 and 8.5 with a pH optimum between 7.75 and 8.0.

Effect of divalent cations on the purified rat dIGnT
The activity of the purified rat dIGnT was not dependent on divalent cations. MnCl2, FeCl2, CoCl2, CuCl2, NiCl2, and ZnCl2 inhibited enzyme activity, whereas CaCl2 and MgCl2 had no substantial inhibitory effects on enzyme activity.

Amino acid sequence analysis
To determine the partial amino acid sequence of the purified rat dIGnT, large-scale purification was carried out as described in the Materials and methods. The purified enzyme was subjected to SDS–PAGE under reducing conditions followed by staining with Coomassie brilliant blue R-250. Two bands of 52 K and 61 K were detected in the gel (data not shown). Each of the two bands was digested with lysylendopeptidase, and the resulting peptides were isolated on a reversed-phase high-performance liquid chromatography column. We successfully obtained partial amino acid sequences for the band of 52 K but not from that of 61 K. There might be some contamination in the 61 K component, which resulted in a poor yield of digested peptides. Amino acid sequences determined for three lysylendopeptidase-digested peptides from the 52 K band are shown in Figure 2. A homology search of each of the three peptides revealed a significant homology with human C2GnT-M (Yeh et al., 1999Go), pBORFF3-4 (a viral ortholog of C2GnT-M; Vanderplasschen et al., 2000Go), and AK008234, a clone of the RIKEN full-length enriched mouse cDNA library (a putative mouse ortholog of C2GnT-M) (Figure 2). To confirm the homology of the rat dIGnT with human and viral C2GnT-Ms over its entire sequence, we decided to clone the cDNA encoding the rat dIGnT.



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Fig. 2. Partial amino acid sequences of the purified rat dIGnT (P123, P133, and P239 + 240). The amino acid sequences are shown using the single-letter code. X indicates an amino acid residue that could not be assigned because of assay difficulties. Capital letters indicate firm assignments, whereas lowercase letters indicate tentative assignments. Amino-terminal lysine residues are estimated from the specificity of the lysylendopeptidase. Homologous sequences of a putative mouse C2GnT-M (AK008234), human C2GnT-M (h-C2GnT-M), and viral C2GnT-M (pBORFF3-4) are aligned. Introduced gaps are shown as hyphens, and aligned identical residues are boxed (black). The numbers indicate the amino acid residue numbers.

 
Isolation of cDNA encoding rat dIGnT
To clone the rat dIGnT cDNA, we utilized the sequence of the clone AK008234. We synthesized sense and antisense primers against both ends of a predicted catalytic domain region of this clone. Using these primers, a corresponding rat cDNA fragment (1188 bp) was successfully amplified by reverse transcription polymerase chain reaction (RT-PCR) on rat small intestinal total RNA. Based on the sequence of the amplified cDNA fragment, 5'- and 3'-rapid amplification of cDNA ends (RACE) were performed on rat small intestinal total RNA to obtain the full coding sequence of the rat cDNA. As shown in Figure 3, the cDNA consisted of 1992 nucleotides having an open reading frame corresponding to 437 amino acid residues with a molecular weight of 50,639. The deduced amino acid sequence included all peptides obtained by amino acid sequencing of the purified rat dIGnT. Two potential N-linked glycosylation sites were included in the deduced amino acid sequence. A Kyte and Doolittle (1983) hydropathy analysis revealed a potential transmembrane domain comprised of 19 amino acids in the amino-terminal region, predicting that this protein has a type II transmembrane topology, as has been the case for the majority of glycosyltransferases cloned to date.



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Fig. 3. Nucleotide and deduced amino acid sequences of rat dIGnT. The predicted amino acid sequence is shown in single-letter code. The positions of the three peptide sequences obtained by digestion of the purified rat dIGnT are single-underscored. Asterisks indicate potential N-glycosylation sites. The putative transmembrane hydrophobic domain is double-underscored. A potential polyadenylation signal is indicated in bold.

 
A homology search revealed a 77% and 74% homology with human C2GnT-M and pBORFF3-4, respectively (Figure 4), indicating that the rat dIGnT is the rat ortholog of human and viral C2GnT-Ms.



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Fig. 4. Alignment of the predicted amino acid sequences of rat dIGnT (r-dIGnT), human C2GnT-M (h-C2GnT-M), viral C2GnT-M (pBORFF3-4), and a putative mouse C2GnT-M (AK008234). Sequence alignment was carried out using a Clustal program. Introduced gaps are shown as hyphens, and aligned identical residues are boxed (black).

 
Acceptor substrate specificity of rat dIGnT
The acceptor substrate specificity of rat dIGnT was examined using four different types of acceptor substrates, lacto-N-triose II-PA, lacto-N-neotetraose-PA, mucin core 1{alpha}-paranitrophenyl (pNP), and mucin core 3{alpha}-pNP by assay method II as described in Materials and methods, because human and viral C2GnT-Ms are known to have broad acceptor substrate specificity (Schwientek et al., 1999Go; Vanderplasschen et al., 2000Go; Yeh et al., 1999Go). The cloned cDNA was inserted into a mammalian expression vector, pSVK3, and overexpressed in COS-1 cells. As shown in Table II, COS-1 cells transfected with the pSV-r-dIGnT showed high activities when lacto-N-triose II-PA, core 1{alpha}-pNP, and core 3{alpha}-pNP were used as acceptor substrates, compared with COS-1 cells transfected with no or control plasmid. This enzyme activity for core 1{alpha}-pNP and core 3{alpha}-pNP was identified to be C2GnT and C4GnT activity by structural elucidation of these products (described in the following section). The ratio of these activities for the pSV-r-dIGnT transfectant was 0.34 (dIGnT), 1.00 (C2GnT), and 0.90 (C4GnT). The purified enzyme preparation also showed almost the same acceptor specificity (Table II), indicating that the cloned enzyme clearly corresponds to the purified rat dIGnT.


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Table II. Acceptor substrate specificity of rat dIGnT

 
The acceptor specificity of dIGnT in the flow-through fraction of lacto-N-triose II-aminocellulofine column chromatography (Figure 1E) was also examined. The result was almost the same as those of the purified and the cloned dIGnTs.

Identification of the enzymatic products
Reactions of the purified dIGnT with mucin core 1 and core 3 analogs
The purified dIGnT effectively catalyzed reactions in which the mucin core 1 and core 3 analogs served as acceptors. Desalted reaction mixtures revealed two characteristic UV-absorbing components in gel filtration experiments (Figures 5B and D), whereas the acceptors revealed only one component (Figures 5A and C). Electrospray ionization (ESI) mass spectrometry (MS) experiments showed that both products had one extra GlcNAc residue compared to the corresponding acceptors (spectrum not shown). The identification of the isolated products was performed by one-dimensional proton nuclear magnetic resonance (1H NMR) spectrometry (Figure 6 and Table III). The J1,2-coupling constants of the H-1 s of the new GlcNAc units in both compounds were around 8.5 Hz, indicating attachment through ß-linkages. A direct comparison of the chemical shifts with data reported for the mucin core 2 {alpha}-pNP glycoside (Schwientek et al., 2000Go) and mucin core 4 {alpha}-pNP glycoside (Ropp et al., 1991Go; Schwientek et al., 1999Go) established that the purified dIGnT generated the mucin core 2 and core 4 sequences from core 1 and core 3 analogs, respectively.



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Fig. 5. Gel filtration experiments in a Superdex Peptide column with water elution at a flow rate of 0.5 ml/min. (A) Core 1{alpha}-pNP; (B) oligosaccharide mixture resulting from incubation of the purified rat dIGnT with core 1{alpha}-pNP and UDP-[14C]GlcNAc; (C) core 3{alpha}-pNP; (D) oligosaccharide mixture resulting from incubation of the purified rat dIGnT with core 3{alpha}-pNP and UDP-[14C]GlcNAc.

 


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Fig. 6. Sections of 1H NMR spectra of the core 2 and core 4 analogs formed in the reactions catalyzed by the purified rat dIGnT. (A) Core 2 analog, Galß1-3(GlcNAcß1-6)GalNAc{alpha}-pNP; (B) Core 4 analog, GlcNAcß1-3(GlcNAcß1-6)GalNAc{alpha}-pNP. Residue designations are as follows: 1, GalNAc; 2, Galß1-3 or GlcNAcß1-3; and 3, GlcNAcß1-6.

 

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Table III. 1H chemical shifts of monosaccharide reporter groups in pNP-glycosides at 23°C

 
Reactions of the purified dIGnT with polylactosamines
The enzymatic products derived from reaction of the purified rat dIGnT with two different oligosaccharides of the polylactosamine type, that is, GlcNAcß1-3'LacNAc and LacNAcß1-3'LacNAc, were characterized. Under the conditions of the standard reaction described in Materials and methods, the enzyme catalyzed the transfer of substantial amounts of [14C]GlcNAc to GlcNAcß1-3'LacNAc. After removal of the surplus donor and other acidic molecules by filtration through a bed of ion exchange resins, the neutral oligosaccharides in the reaction mixture were subjected to matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS), which revealed the presence of molecular ions of the product (GlcNAc)2-LacNAc and the acceptor GlcNAc-LacNAc in a molar ratio of 10:90. The product was purified by gel filtration chromatography and then subjected to one-dimensional 1H-NMR spectroscopy. The spectrum (not shown) established that the product was the tetrasaccharide GlcNAcß1-3'(GlcNAcß1-6')LacNAc. In particular, the presence of a doublet of one equivalent at 4.585 ppm (J1,2 = 8.5Hz) is a highly characteristic resonance of the H1 of a GlcNAc, when it is linked to galactose via a ß1,6-linkage. All the other reporter groups of the product tetrasaccharide gave signals that were identical to those reported previously for this tetrasaccharide (Koenderman et al., 1987Go; Renkonen et al., 1991aGo).

A parallel transferase experiment with the tetrasaccharide LacNAcß1-3'LacNAc as the acceptor generated no measurable net radioactivity to the resulting fraction of neutral oligosaccharides. In addition, MALDI-TOF MS of this fraction failed to reveal the presence of the pentasaccharide product LacNAcß1-3'(GlcNAcß1-6')LacNAc (M + Na+ calculated m/z 974.4); only the molecular ions (M + Na)+ and (M + K)+ of the acceptor LacNAcß1-3'LacNAc were observed in the molecular ion range of the spectrum (data not shown). These data suggest that the purified dIGnT did not react with the tetrasaccharide LacNAcß1-3'LacNAc at a significant rate, showing that it lacks cIGnT activity.


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 Materials and methods
 References
 
ß1-6GnTs are a family of enzymes that are capable of generating four different GlcNAcß1,6 linkages (in bold): GlcNAcß1-3(GlcNAcß1-6)Gal (blood group I-type structure), Galß1-3(GlcNAcß1-6)GalNAc (mucin core 2 structure), GlcNAcß1-3(GlcNAcß1-6)GalNAc (mucin core 4 structure), and GlcNAcß1-2(GlcNAcß1-6)Man (2,6-branched N-linked core structure) (Bierhuizen and Fukuda, 1994Go; van den Eijnden and Joziasse, 1993Go). Thus, ß1-6GnTs can be classified into four major groups dependent on their acceptor specificities: blood group I GnT, C2GnT, C4GnT, and GnT V. All the ß1-6GnTs that have been characterized to date have no requirement for the Mn2+ ion, in contrast to other GnTs, which require divalent metal ions for full activity. Among the IGnTs, two types of enzymes, that is, dIGnT and cIGnT, are known. The distally acting enzyme (dIGnT) acts on a subterminal Gal residue that is already substituted at position 3 by a GlcNAc residue. In contrast, the centrally acting enzyme (cIGnT) transfers a GlcNAc unit to a midchain Gal residue that is already substituted at position 3 by LacNAc units. To date, three cIGnTs have been cloned (Bierhuizen et al., 1993Go; Chen et al., 2000Go) or purified (Sakamoto et al., 1998Go), and none have been found to contain substantial dIGnT activity (Sakamoto et al., 1998Go; Mattila et al., 1998Go; Chen et al., 2000Go; Ujita et al., 1999Go).

Thus far, several publications (Ropp et al., 1991Go; Schwientek et al., 1999Go; Vanderplasschen et al., 2000Go; Yeh et al., 1999Go) concerning proteins having dIGnT activity have appeared. Bovine tracheal dIGnT activity was purified to apparent homogeneity, but a lack of purified material prevented its cDNA cloning (Ropp et al., 1991Go). Apart from this purification approach, two groups recently reported that the human homolog of C2GnT-L (leukocyte-type C2GnT; Bierhuizen and Fukuda, 1992Go) had dIGnT activity as well as C4GnT activity (Schwientek et al., 1999Go; Yeh et al., 1999Go), whereas C2GnT-L possesses neither of these two activities. This newly identified homolog was named mucin-type C2GnT (C2GnT-M) because it is mainly expressed in mucin producing tissues at the mRNA level (Yeh et al., 1999Go). An ortholog of C2GnT-M was identified in bovine herpes virus type 4, which was also shown to have dIGnT and C4GnT activities (Vanderplasschen et al., 2000Go). The relationship between purified bovine tracheal dIGnT and C2GnT-M is not clear, although bovine tracheal dIGnT also possesses C2/C4GnT activities.

In this study, we have purified the dIGnT to apparent homogeneity from rat small intestine by column chromatographic procedures using Q-Sepharose FF, Ni2+-chelating Sepharose FF, Zn2+-chelating Sepharose FF, UDP-hexanolamine-agarose, and lacto-N-triose II-aminocellulofine, using an assay wherein pyridylaminated lacto-N-triose II (GlcNAcß1-3Galß1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcß1-3(GlcNAcß1-6)Galß1-4Glc-PA. The purified enzyme has an apparent molecular weight of 66,000 under nonreducing conditions. dIGnT activity is extremely unstable at steps 2 to 3. This might provide a partial explanation for why the purification-fold does not increase significantly up to step 3 (Table I). This instability in dIGnT activity was also noted by Ropp et al. (1991)Go. In their study, Galß1-3GalNAc{alpha}-benzyl was added during the Triton X-100 solubilization, which has proven to be effective for stabilizing enzyme activity. However, for such a large-scale enzyme preparation as reported herein, we were unable to include this sugar substrate during the purification process. Thus, a reduction in the time required for purification became critical. Steps 2 to 3 should be done within 10 h. At the Q-Sepharose FF column chromatography step, a column with a flattened shape (i.e., wide radius) should be used to get a decent speed of the fractionation (750 ml/h). After we established the complete purification protocol using retired rats, we found that small intestine from young rats (8 weeks old) was a better source than retired rats in terms of the yield of enzyme activity. Although the total enzyme activity per weight and specific enzyme activity found in small intestine is essentially the same, the yield of dIGnT activity from young rats after Triton X-100 extraction step was found to be about fivefold higher than that obtained from old rats. The possible difference in the lipid composition in the microsomal fraction might explain this observation. For this reason, we used small intestine of young rats for large scale preparation.

The analysis of acceptor substrate specificity of the purified rat dIGnT revealed that it also has other enzyme activities. It is capable of effectively adding a ß1,6GlcNAc branch to mucin core 1 and core 3 analogs as well as to the peridistal galactoses (in bold) in polylactosamine type substrate GlcNAcß1-3Galß1-4GlcNAc, showing that this enzyme has C2GnT and C4GnT activities as well as dIGnT activity. This broad acceptor substrate specificity seems to be typical among enzymes which have dIGnT activity.

We have cloned a cDNA encoding rat dIGnT based on the partial amino acid sequences of the purified enzyme. Several lines of evidence indicate that the cloned cDNA corresponds to the purified rat dIGnT: (a) The deduced protein sequence contains all three peptides of the purified enzyme; (b) when the cDNA was transiently expressed in COS-1 cells, the enzyme activities were highly expressed; and (c) the cDNA-transfected COS-1 cells showed the same acceptor substrate specificity as were found for the purified enzyme preparation. The deduced amino acid sequence of rat dIGnT shares 77% and 74% homology with those of human C2GnT-M (Yeh et al., 1999Go) and pBORFF3-4 (a viral ortholog of C2GnT-M; Vanderplasschen et al., 2000Go), respectively. This indicates that the cloned rat dIGnT is the orthologous gene product of human and viral C2GnT-Ms. However, the acceptor substrate specificity of rat dIGnT differs from that reported for human and viral C2GnT-Ms. As shown in Table IV, rat dIGnT enzyme has higher apparent dI/C4GnT activities than human and viral C2GnT-Ms. It is particularly noteworthy that rat dIGnT has the comparable amount of C4GnT activity (0.90) to C2GnT activity (1.00). This difference in acceptor substrate specificity might be attributable to a species-specific difference. Some of the unconserved amino acid residues among the three orthologous enzymes in a putative catalytic domain could contribute to their various substrate specificities. We found that a putative mouse ortholog of C2GnT-M (AK008234) has the highest sequence similarity to the rat dIGnT (Figure 4). It would be the case that the mouse enzyme shows acceptor substrate specificity similar to rat dIGnT. Because purified bovine tracheal dIGnT shows almost the same acceptor substrate specificity (Ropp et al., 1991Go; Table IV) as rat dIGnT, it is also likely that this bovine enzyme is an orthologous gene product of rat dIGnT.


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Table IV. Summary of acceptor substrate specificity for rat dIGnT and other known C2GnTs

 
We could not detect cIGnT activity in the rat dIGnT (Table II), although human and viral C2GnT-Ms are reported to have marginal but detectable cIGnT activity (around 3% compared to C2GnT activity) (Table IV). Currently, it is not certain if it is attributable to a species-specific difference, or if our assay is not sensitive enough to detect such a very minor activity.

In summary, the present data indicate this dIGnT purified from rat small intestine is the orthologous gene product of human and viral C2GnT-Ms. Because there is no any other protein band visible except this protein in the fraction which contains highly concentrated dIGnT activity (see Figure 1E and F, fraction number 18), it should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue. Mucin core 2 and core 4 structures in rat small intestine could also be mainly formed by the function of this enzyme, given the fact that this enzyme has strong C2 and C4GnT activities compared with dIGnT activity. To confirm this possibility, an assay for C2/C4GnT activities throughout our purification process would be necessary.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials were obtained from the following suppliers: UDP-GlcNAc, UDP-hexanolamine-agarose (ligand concentration: 2.1 µmol/ml), and bovine testis ß-galactosidase from Sigma (St. Louis, MO); Q-Sepharose FF, chelating Sepharose FF, Superdex Peptide HR 10/30, and UDP-[14C]GlcNAc from Amersham Pharmacia Biotech (Uppsala, Sweden); aminocellulofine, lacto-N-tetraose, lacto-N-neotetraose, and lacto-N-hexaose from Seikagaku (Tokyo); Dowex AG1, Dowex AG50, and Bio-Gel P-4 from Bio-Rad Laboratories (Hercules, CA); Galß1-3GalNAc{alpha}1-O-pNP (mucin core 1 analog) and GlcNAcß1-3GalNAc{alpha}1-O-pNP (mucin core 3 analog) from Toronto Research Chemicals (North York, ON). The purity of these core 1 and 3 analogs were verified by their ESI mass spectra, showing major signals having the expected m/z values (not shown). Relevant NMR data are shown in Table III. Other common chemicals were obtained from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

Preparation of pyridylaminated substrates and the authentic product
Galß1-4GlcNAcß1-3Galß1-4Glc-PA (lacto-N-neotetraose-PA), Galß1-3GlcNAcß1-3Galß1-4Glc-PA (lacto-N-tetraose-PA), and Galß1-3GlcNAcß1-3(Galß1-4GlcNAcß1-6)Galß1-4Glc-PA (lacto-N-hexaose-PA) were prepared by the pyridylamination of lacto-N-neotetraose, lacto-N-tetraose, and lacto-N-hexaose, respectively, using Glyco TAG (Takara, Shiga, Japan), a commercially available pyridylamination apparatus. GlcNAcß1-3Galß1-4Glc-PA (lacto-N-triose II-PA) and GlcNAcß1-3(GlcNAcß1-6) Galß1-4Glc-PA were prepared by digestion of lacto-N-tetraose-PA and of lacto-N-hexaose-PA, respectively, with bovine testis ß-galactosidase.

Preparation of polylactosamine substrates
GlcNAcß1-3Galß1-4GlcNAc was synthesized as described previously (Seppo et al., 1990Go). The MALDI-TOF mass spectrum of this product revealed a major signal in the range of the molecular ions at m/z 609.2; it was assigned to (M + Na)+ for GlcNAcß1-3Galß1-4GlcNAc (calculated m/z 609.2). The NMR spectrum was in agreement with previously reported data (Koenderman et al., 1987Go), except that the H-1 doublet of the nonreducing end GlcNAc in our sample appeared at 4.684 ppm in the {alpha}-form of the saccharide and at 4.680 ppm in the ß-form, whereas the corresponding values in the reference (Koenderman et al., 1987Go) were reported to be 4.689 and 4.684 ppm, respectively. No resonances were observed in our spectrum in the 4.58–4.59 ppm region, corresponding to the area of H-1 signals of ß1,6-bonded GlcNAc residues (Helin et al., 1997Go; Leppänen et al., 1997bGo).

Galß1-4GlcNAcß1-3Galß1-4GlcNAc was synthesized enzymatically by ß4-galactosylating GlcNAcß1-3Galß1-4GlcNAc as described previously (Renkonen et al., 1991bGo). The MALDI-TOF mass spectrum of the sample revealed a major signal in the range of molecular ions at m/z 771.3; it was assigned to (M + Na)+ of Galß1-4GlcNAcß1-3Galß1-4GlcNAc (calculated m/z 771.3). The 1H-NMR spectrum was identical to that of Galß1-4GlcNAcß1-3Galß1-4GlcNAc reported previously (Maaheimo et al., 1997Go).

Preparation of lacto-N-triose II-aminocellulofine resin
Lacto-N-tetraose (7 µmol) was digested with 100 mU of bovine testis ß-galactosidase for 48 h at 37°C in 2.7 ml of 5 mM citrate phosphate buffer (pH 4.0). After boiling for 3 min, followed by centrifugation at 15,000 rpm for 15 min, 0.9-ml aliquots of the supernatant were loaded on a Toyopearl HW-40F column (1x25 cm; Tosoh, Tokyo), which had been equilibrated with 5% ethanol. This column chromatographic procedure was repeated three times. The eluent was monitored by MALDI-TOF MS, and the fractions containing lacto-N-triose II were combined and lyophilized. Lacto-N-triose II-aminocellulofine was prepared by coupling lacto-N-triose II (about 6 µmol) and aminocellulofine (0.6 ml) in the presence of NaBH3CN according to the manufacturer's instructions. Nonreacted amino groups were blocked by N-acetylation with acetic anhydride.

Determination of dIGnT activity
Method I
dIGnT activity was assayed according to the method of Gu et al. (1992)Go with minor modifications. The standard assay mixtures contained the following components in a total volume of 10 µl; 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0), 5 mM ethylenediamine tetra-acetic acid (EDTA), 0.5% Triton X-100, 10 mM UDP-GlcNAc, 10 µM lacto-N-triose II-PA, 20 mM GlcNAc, 2 mg/ml BSA, and enzyme fraction (3 µl). After incubation at 37°C for 1–4 h, 40 µl water was added, and the enzyme reaction was terminated by boiling for 1 min. After centrifugation at 12,000 rpm for 5 min, 10 µl of the supernatant from the reaction mixture was applied to two coupled TSK-gel ODS-80TM columns (4.6x250 mm; Tosoh). The elution was performed at 50°C using 20 mM ammonium acetate (pH 4.0) as the eluent, at a flow rate of 1.2 ml/min. Fluorescence was monitored using excitation and emission wavelengths of 320 and 400 nm, respectively. The specific activity of the enzyme was expressed as pmol of product per h per mg protein.

cIGnT activity was assayed at the same conditions as for dIGnT except that the acceptor substrate used was lacto-N-neotetraose-PA.

Method II
Transferase activities were assayed in reaction mixtures that contained the following components in a total volume of 24 µl: 50 mM MOPS buffer (pH 7.0), 10 mM EDTA, 0.5% Triton X-100, 1 mM UDP-[14C]GlcNAc (5200 cpm/nmol), 0.5 mM acceptor substrate, 100 mM GlcNAc, 2 mg/ml BSA, and enzyme source (7.5 µl). After incubation at 37°C for 1 h, the reaction was terminated by dilution with 0.5 ml ice-cold water. The reaction mixture was then passed through a column of AG1-X8 (AcO- form, 0.5 ml) to remove unreacted UDP-[14C]GlcNAc. The column was washed with 2.5 ml of water, and the amount of [14C]GlcNAc incorporated into the acceptor was determined by counting aliquots of the effluent with a liquid scintillation counter. Background radioactivity (derived from the incubation mixtures without acceptor substrate) was subtracted. The specific activity of the enzyme was expressed as nanomoles of product per h per mg of protein.

The protein concentration was determined with a BCA kit (Pierce, Rockford, IL) or a Bio-Rad Protein assay reagent using BSA as the standard.

Buffers used in purification of dIGnT
The buffers used in this study were as follows, with the pH measured at 4°C: buffer A, 0.25 M sucrose, 20 µM ( p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 10 mM Tris–HCl (pH 7.4); buffer B, 20% glycerol, 20 µM ( p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 1% Triton X-100, 10 mM Tris–HCl (pH 7.4); buffer C, 20% glycerol, 0.1% Triton X-100, 20 mM triethanolamine (pH 7.8); buffer D, 20% glycerol, 0.1% Triton X-100, 0.5 M NaCl, 20 mM Tris–HCl (pH 8.0); buffer E, 20% glycerol, 0.1% Triton X-100, 10 mM EDTA, 0.1 M NaCl, 20 mM MOPS (pH 7.0); buffer F, 20% glycerol, 0.1% Triton X-100, 10 mM EDTA, 50 mM NaCl, 20 mM MOPS (pH 7.0); and buffer G, 20% glycerol, 0.1% Triton X-100, 50 mM NaCl, 20 mM MOPS (pH 7.0).

Homogenization and preparation of the microsome fraction (step 1)
All purification steps were carried out at 4°C. Small intestines of 20 retired Sprague Dawley rats (140 g) were homogenized with a Waring blender in three volumes of buffer A. After centrifugation at 8000 rpm for 10 min, the supernatants were pooled, and the pellet was subjected to two more extractions, after which all the supernatants (1300 ml) were combined. After centrifugation at 78,000xg for 2 h, the microsomal fraction (about 30 g) was obtained as a precipitate.

Solubilization of dIGnT (step 2)
The microsomal fraction was suspended in 150 ml of buffer B, gently stirred for 1 h, and then centrifuged at 105,000xg for 1 h. The supernatant fraction was collected, and the residual pellet was subjected to a second extraction and ultracentrifugation. The first and the second Triton X-100 extracts were combined and used for further enzyme purification.

Q-Sepharose FF column chromatography (step 3)
Two volumes of buffer C were added to the Triton X-100 extracts and then applied to a column of Q-Sepharose FF (10x3.8 cm), which had been equilibrated with buffer C. Fractions of 50 ml were collected during this column chromatography. The column was washed with buffer C until the protein concentration was reduced to 0.2 mg/ml. The elution was carried out with a linear gradient established between 900 ml buffer C and 900 ml 0.8 M NaCl/buffer C. Fractions containing dIGnT activity were combined.

Ni2+-chelating Sepharose FF column chromatography (step 4)
An equal volume of Buffer D was added to the pooled enzyme fraction from Step 3 and then applied to a column of Ni2+-chelating Sepharose FF (2.5x6 cm), which had been equilibrated with Buffer D. Ni2+-chelating Sepharose FF resin was layered on the chelating Sepharose FF resin without metal ions (2.5x3.5 cm), to avoid any possible leakage of Ni2+ into the enzyme fractions. Fractions of 6.5 ml were collected during this column chromatography. After washing the column with Buffer D until the protein concentration was reduced to 0.1 mg/ml, dIGnT activity was eluted with a linear gradient established between 200 ml of Buffer D and 200 ml of 0.2 M glycine/Buffer D. Fractions containing dIGnT activity were combined, and then concentrated to 12 ml with an Amicon Diaflow Ultrafiltrater (Millipore, Bedford, MA) using a YM 30 membrane (Millipore).

Zn2+-chelating Sepharose FF column chromatography (step 5)
The concentrated enzyme fraction from step 4 was diluted 10-fold with buffer D and then applied to a Zn2+-chelating Sepharose FF column (1.5x6.8 cm), which had been equilibrated with buffer D. Zn2+-chelating Sepharose FF resin was layered on the chelating Sepharose FF resin without metal ions (1.5x4 cm) to avoid any possible leakage of Zn2+ into the enzyme fractions. Fractions of 3.5 ml were collected during this column chromatography. The column was washed with 100 ml buffer D, and then dIGnT activity was eluted with a linear gradient established between 100 ml buffer D and 100 ml 0.2 M glycine/buffer D. Fractions containing dIGnT activity were combined, and the buffer in this fraction was replaced by buffer E by means of an Amicon Diaflow Ultrafiltrater using a YM 30 membrane.

UDP-hexanolamine-agarose column chromatography (step 6)
After the previous step, the column was siliconized with Sigmacote (Sigma), and siliconized tubes (Assist, Tokyo) were used in the fractionation procedures. The concentrated enzyme fraction from step 5 was applied to a column of UDP-hexanolamine-agarose (1x13 cm), which had been equilibrated with buffer E. Fractions of 1 ml were collected during this column chromatography. After washing with 50 ml buffer E and 30 ml buffer F, dIGnT activity was eluted with a linear gradient established between 30 ml buffer F and 30 ml 2.5 mM UDP/buffer F. Fractions containing dIGnT activity were combined.

Lacto-N-triose II-aminocellulofine column chromatography (step 7)
The pooled enzyme fraction from step 6 was applied directly to a lacto-N-triose II-aminocellulofine column (0.8x 1.2 cm), which had been equilibrated with buffer F. Fractions of 0.6 ml were collected during this column chromatography. After washing with 3 ml buffer F and 3 ml buffer G, dIGnT activity was eluted with 3.6 ml 0.1 M NaCl/buffer G, 3 ml 0.2 M NaCl/buffer G, and 3 ml 0.4 M NaCl/buffer G. The purified enzyme fractions eluted with 0.1 M NaCl/buffer G were pooled and used in the enzyme characterization.

Large scale preparation of the purified rat dIGnT
For a large-scale preparation of the purified rat dIGnT, the purification procedure described was scaled up about twofold, using 260 g of small intestines of 50 young 8-week-old Sprague Dawley rats at one time. This entire purification procedure was repeated three times, and all fractions that contained enzyme activity were pooled. The pooled enzyme fraction was concentrated to about 0.05 ml with a small column of Ni2+-chelating Sepharose FF (7 x 5.5 mm) and a Centricon YM-30 (Millipore). Finally, about 1.5 µg of purified rat dIGnT was obtained from 780 g of rat small intestine in 0.5% yield.

Gel electrophoresis
SDS–PAGE was performed by the method of Laemmli (1970)Go using 8.5% gels. Molecular markers (Amersham Pharmacia Biotech) were used for size standards. Proteins in the gels were stained by means of a silver staining kit (2D silver stain II "DAIICHI"; Daiichi Pure Chemicals, Tokyo).

Amino acid sequencing of peptides derived from purified rat dIGnT
About 0.9 µg of purified rat dIGnT was subjected to SDS–PAGE under reducing conditions followed by staining with Coomassie brilliant blue R-250. Two bands corresponding to 52 K and 61 K were detected. Each of the two bands was excised and treated with 0.1 µg of Achromobacter protease I (lysylendopeptidase) (Masaki et al., 1981Go) (a gift from Dr. T. Masaki, Ibaraki University, Ibaraki, Japan) at 37°C overnight in 0.1 M Tris–HCl (pH 9.0) containing 0.1% SDS and 1 mM EDTA. The generated peptides were extracted from the gel and separated on columns of DEAE-5PW (1x20 mm; Tosoh) and Mightysil RP-18 (1x50 mm; Kanto Chemicals, Tokyo) connected in series with a model 1100 (Hewlett Packard) liquid chromatography system. The peptides were eluted at a flow rate of 20 µl/min using an elution program of 1–12.5–50% solvent B in 0–10–70 min, where solvents A and B were 0.085% (v/v) aqueous trifluoroacetic acid and 0.075% trifluoroacetic acid in 80% (v/v) acetonitrile, respectively. Selected peptides were subjected to Edman degradation using a model Procise 494 cLC sequencer (Applied Biosystems, Foster City, CA) and to MALDI-TOF MS (Bruker-Franzen Analytic, Breman, Germany) in the linear mode using 2-mercaptobenzothiazol (Xu et al., 1997Go) as the matrix.

Isolation of cDNA encoding rat dIGnT
Through BLASTp analysis of the obtained partial amino acid sequences of the purified enzyme, AK008234, a clone of the RIKEN full-length enriched mouse cDNA library (a putative mouse ortholog of human and viral C2GnT-Ms; Yeh et al., 1999Go; Vanderplasschen et al., 2000Go) was identified. We utilized the sequence of the clone AK008234 to obtain rat dIGnT cDNA. We synthesized oligonucleotide primers, the sense primer RHS1 (5'-ACTCTGAGGAATTTCAAAGC-3'), and the antisense primer RHA2 (5'-CTCATAGTTCAGTCCCATAG-3') against both ends of a predicted catalytic domain region of this clone. Using these primer sets, the corresponding rat cDNA fragment was amplified by RT-PCR (30 cycles at 95°C for 30 s; 50°C for 30 s; 72°C for 1 min 15 s) on rat small intestinal total RNA. The RT-PCR product (1188 bp) was subcloned into the pGEM-T Easy vector (Promega, Madison, WI).

Based on the sequence of the RT-PCR product, 5'- and 3'-RACE were performed to obtain the full coding sequence of the rat cDNA. Concerning 5'-RACE, rat small intestinal total RNA was reverse-transcribed with the antisense primer RHA8 (5'-AAGGTGCTCATCAGGAC-3'), and a poly(dA) tail was added with a terminal deoxynucleotidyltransferase (Life Technologies, Grand Island, NY). The primary amplification was performed by PCR (25 cycles at 95°C for 30 s, 50°C for 30 s, 72°C for 1 min 30 s) using the adapter primer (Life Technology) and the antisense primer RHA3 (5'-AGTCTTCCATGCAGTTC-3'). In the secondary amplification, PCR (25 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 1 min) was performed using the abridged universal amplification primer (Life Technology) and the nested antisense primer RHA6 (5'-CAGCCTCTG-TGAAGGACTGC-3'). Concerning 3'-RACE, rat small intestinal total RNA was reverse-transcribed with adapter primer, and PCR (25 cycles at 95°C for 30 s, 58°C for 30 s, 72°C for 1 min) was performed using the sense primer RHS4 (5'-GAGGGAGACATTGAGAATGG-3') and the abridged universal amplification primer. The 5'- and 3'-RACE products were subcloned into the pGEM-T Easy vector.

Based on the obtained sequence, cDNA encoding the rat dIGnT was amplified by RT-PCR (30 cycles at 95°C for 30 s, 50°C for 30 s, 72°C for 3 min) on rat small intestinal total RNA using a Pfu DNA polymerase (Promega) with the sense primer RHS6 (5'-GATGGTTCCCACCTGTGA-AG-3') and the antisense primer RHA10 (5'-ATCATCT-TGCAGTGAGTCTC-3'). The cDNA obtained by RT-PCR was subcloned into EcoRV site of the pBluescript II SK vector (Stratagene, La Jolla, CA) (termed pBS- r-dIGnT).

DNA sequencing
The subcloned cDNAs were sequenced by the dideoxy chain termination method (Big Dye Terminator cycle sequencing kit, Applied Biosystems) using an ABI PRISM model 3100 DNA sequencer (Applied Biosystems).

Expression of cDNA encoding rat dIGnT in COS-1 cells
pBS-r-dIGnT was digested with EcoRI and SalI, and the inserted DNA was ligated between the EcoRI and SalI sites of the expression vector pSVK3 (Amersham Pharmacia Biotech) (termed pSV-r-dIGnT). COS-1 cells (5x106) suspended in 0.8 ml transfection buffer, pH 7.4, 30 mM NaCl, 120 mM KCl, 5 mM MgCl2, 8 mM Na2HPO4, 1.5 mM KH2PO4, were transiently transfected with 20 µg plasmid DNA by electroporation (220 V, 960 µF). Two days after transfection, the cells were harvested and sonicated with 0.1 ml of ice-cold phosphate-buffered saline. Enzyme activity was assayed using this cell lysate as the enzyme source.

Large preparation of the enzymatic products
The acceptors (100 nmol) and donors, representing a mixture of UDP-GlcNAc (250 nmol) and UDP-[14C]GlcNAc (500,000 cpm), were incubated with the purified rat dIGnT fraction (12.5 µl) at 37°C for 25 h in a total volume of 25 µl containing 50 mM MOPS (pH 7.5), 5 mM EDTA, 75 mM NaCl, 20 mM GlcNAc, 20 mM Gal, 8 mM NaN3, 0.5% Triton X-100, 10% glycerol, and 2 mg/ml BSA. The reactions were terminated by heating in a boiling water bath for 5 min. Control reactions were performed without exogenous acceptors. The transferase reaction mixtures were then passed in water through a mixed bed of Dowex AG1 (AcO-) and Dowex AG50 (H+) to remove salts and unreacted UDP-[14C]GlcNAc. The amounts of [14C]GlcNAc incorporated into the acceptors were then determined from aliquots of the filtrate by liquid scintillation counting as described previously (Leppänen et al., 1997aGo).

Further purification of the products was achieved by gel permeation chromatography on a single or two coupled Superdex Peptide HR 10/30 columns (equilibrated with water, flow rate of 0.5 or 1.0 ml/min; the runs of oligo- N-acetyllactosamines were monitored at 214 nm and those of para-nitrophenyl glycosides at 270 nm) or on a Bio-Gel P-4 column (200–400 mesh, 1x145 cm, equilibrated with water, monitored by UV absorption at 205 nm and by liquid scintillation counting).

NMR spectroscopy
One-dimensional 1H NMR spectra of the acceptor saccharides were recorded at 23°C in 600 µl of D2O at 500 MHz on a Varian Unity 500 spectrometer (Palo Alto, CA), as described previously (Leppänen et al., 1997bGo). 1H NMR spectra of the product saccharides were obtained by dissolving the samples in 39.5 µl of D2O and recording the spectra at 23°C at 500 MHz on a Varian Unity 500 spectrometer equipped with a microcoil probe.

MALDI-TOF and ESI MS of enzymatic products
MALDI-TOF MS was performed in the positive-ion delayed extraction mode with a BIFLEXTM mass spectrometer (Bruker-Franzen Analytik), using a 337 nm nitrogen laser. One microliter of aqueous sample solution (20 pmol) and 1.0 µl of 2,5-dihydroxybenzoic acid matrix (10 mg/ml in water) were mixed on the target plate and dried under a gentle stream of air. Maltohexaose and N-acetylgalactosaminodecamer were used for external calibration.

ESI mass spectra were collected in the positive ion mode using a Q-TOF hybrid quadrupole-orthogonal acceleration time-of-flight mass spectrometer (Micromass, U.K.). Samples were dissolved in 0.5 mM aqueous NaOH/acetonitrile (1:1, by volume), and injected into the mass spectrometer with a nanoelectrospray ion source.


    Acknowledgements
 
This work was supported in part by Grant-in-Aid for Scientific Research (S) no. 13854010 from the Japan Society for the Promotion of Science. We thank Dr. Ritva Niemelä for an early MALDI-TOF MS contribution and Olli Aitio for assistance in NMR spectroscopy. We thank Dr. Milton S. Feather (Scientific Editorial Services) for correcting this manuscript. The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank/EBI Data Bank with accession number AB098520.


    Footnotes
 
1 Present address: High Throughput Factory, RIKEN Harima Institute 1-1-1, Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan Back

2 To whom correspondence should be addressed; e-mail: proftani{at}biochem.med.osaka-u.ac.jp Back


    Abbreviations
 
BSA, bovine serum albumin; IGnT, I-branching ß1, 6N-acetylglucoaminyltransferase; C2GnT, core 2 ß1, 6N-acetylglucoaminyltransferase; C4GnT, core 4 ß1,6N-acetylglucoaminyltransferase; EDTA, ethylenediamine tetra-acetic acid; ESI, electrospray ionization; GnT, N-acetylglucoaminyltransferase; 1H NMR, proton nuclear magnetic resonance; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MOPS, 3-(N-morpholino)propanesulfonic acid; PA, 2-aminopyridine; PCR, polymerase chain reaction; pNP, paranitrophenyl; RACE, rapid amplification of cDNA ends; RT, reverse transcription; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Bierhuizen, M.F.A. and Fukuda, M. (1992) Expression cloning of a cDNA encoding UDP-GlcNAc:Galß1-3-GalNAc-R (GlcNAc to GalNAc) ß1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen. Proc. Natl. Acad. Sci. USA, 89, 9326–9330.[Abstract]

Bierhuizen, M.F.A. and Fukuda, M. (1994) ß1,6N-acetylglucosaminyltransferase: enzymes critically involved in oligosaccharide branching. Trends Glycosci. Glycotech., 6, 17–28.

Bierhuizen, M.F.A., Mattei, M.-G., and Fukuda, M. (1993) Expression of the developmental I antigen by a cloned human cDNA encoding a member of a ß-1,6-N-acetylglucosaminyltransferase gene family. Genes Dev., 7, 468–478.[Abstract]

Brockhausen, I., Matta, K.L., Orr, J., Schachter, H., Koenderman, A.H.L., and van den Eijnden, D.H. (1986) Mucin synthesis. Conversion of R1-ß1-3Gal-R2 to R1-ß1-3(GlcNAcß1-6)Gal-R2 and of R1-ß1-3GalNAc-R2 to R1-ß1-3(GlcNAcß1-6)GalNAc-R2 by a ß6-N-acetylglucosaminyltransferase in pig gastric mucosa. Eur. J. Biochem., 157, 463–474.[Abstract]

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