cDNA Cloning and Expression of Bovine UDP-N-Acetylglucosamine:alpha 1,3-D-Mannoside beta 1,4-N-Acetylglucosaminyltransferase IV*

Mari Toba MinowaDagger , Suguru Oguri§, Aruto Yoshida, Tomoka Hara, Akihiro Iwamatsu, Hiroshi Ikenaga, and Makoto Takeuchi

From the Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fuku-ura, Kanazawa-ku, Yokohama, 236-0004, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

UDP-N-acetylglucosamine:alpha 1,3-D-mannoside beta 1,4-N-acetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri- and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases. N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

UDP-N-acetylglucosamine:alpha 1,3-D-mannoside beta 1,4-N-acetylglucosaminyltransferase (GnT-IV)1 (EC 2.4.1.145) is one of the essential enzymes in the synthesis of the multiantennary structure of Asn-linked sugar chains. It specifically generates the beta 1-4 linkage of GlcNAc to the alpha 1-3 Man of the core structure of N-glycans (Man3GlcNAc2). GnT-IV produces tetra-antennary structures in cooperation with GnT-V, which produces the beta 1-6 linkage of GlcNAc to the alpha 1-6 Man of the core structure. This tetra-antennary structure is dominant in certain glycoproteins, such as erythropoietin (EPO) and alpha 1-acid glycoprotein, and very important for their bioactivity (1, 2). For instance, the in vivo biological activity of recombinant EPO has a positive correlation with the ratio of tetra-/biantennary sugar chains. Pharmacokinetics studies have shown that the bulky structure of the tetra-antennary branching prevents EPO from filtering out into the urine. The increase in the antenna number of alpha 1-acid glycoprotein observed under a chronic inflammatory state or liver damage suggested that some clinical situations and/or cellular disorders may also affect the branch formation of sugar chains (3). On the other hand, structural analyses of oligosaccharides in hepatoma cell lines and in choriocarcinoma have suggested that GnT-IV activity should be high in certain kinds of tumors (4-7). Rat 3Y1 cells transformed with the adenovirus 12 gene also elevated the expression of the beta 1-4 GlcNAc on the alpha 1-3 Man (8).

To date, GnT-I, -II, -III, and -V have been purified and their cDNAs and/or genomic DNAs have been cloned (9-23). Among the enzymes necessary to produce tetra-antennary sugar chains, only GnT-IV had not been isolated nor its gene cloned. Due to the lack of data concerning GnT-IV, the mechanism of branch formation has not been fully clarified. Recently Oguri et al. (24) successfully purified and characterized GnT-IV from the bovine small intestine. In this report, we describe the amino acid sequencing of the purified GnT-IV enzyme and cDNA cloning of the bovine GnT-IV gene. We have also localized the catalytic domain of GnT-IV activity and examined its transcription level in a variety of bovine tissues.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Amino Acid Sequencing and Primers for PCR-- GnT-IV was purified from bovine small intestine as described in our previous report (24). The amino acid sequences of protease-digested fragments of the enzyme were determined by Iwamatsu's method (25). Briefly, after the second affinity column chromatography step, 300 pmol of the enzyme preparation was separated on 10% SDS-polyacrylamide gel electrophoresis. After transfer to a polyvinylidene difluoride membrane (ProBlott, Perkin-Elmer), the GnT-IV band was identified by staining with Ponceau S (Sigma) and cut out. The enzyme on the membrane was reduced and S-carboxymethylated to allow detection of Cys residues by the peptide sequencer. The peptide was then digested with the lysil endopeptidase Achromobacter protease I (Wako Pure Chemicals, Osaka, Japan), and the fragments released (AP fragments) were collected. The peptide remaining on the membrane was further incubated with asparagil endopeptidase Asp-N (Boehringer Mannheim), and the digests (DN fragments) were collected. Each peptide fragment was separated by reversed phase high performance liquid chromatography (Wakosil-II AR 5C18 2.0 × 150 mm, Wako Pure Chemicals) and analyzed with a gas-phase peptide sequencer (PPSQ-10, Shimadzu). To determine the N terminus of the purified bovine GnT-IV, 120 pmol of the final enzyme preparation, after the gel filtration, was directly analyzed by the peptide sequencer (24). A bovine codon usage table was constructed using bovine open reading frames randomly selected from the GenBankTM data base. Only codons frequently used for bovine cDNA were adopted to design primers and probes to minimize the variation of primers and probes to be less than 10,000. The primers and probes were designed to be greater than 29- and 41-mer in length, respectively.

The Use of RT-PCR to Isolate a Probe for Library Screening-- Bovine small intestine mRNA was prepared by the guanidinium thiocyanate/CsCl method (26) and a Poly(A) Quik mRNA isolation kit (Stratagene). Sense and antisense primers and probes were synthesized for the AP-5, AP-12, AP-15, and DN-9 peptide fragments (Table I). One of the possible combinations of the sense/antisense primers (50 pmol each) shown in Table I and 0.5 µg of poly(A)+ RNA were used for RT-PCR with an Access RT-PCR kit (Promega). The temperature cycle for RT-PCR was 48 °C for 45 min, 94 °C for 2 min, followed by 50 cycles of 94 °C for 30 s, 60 °C for 1 min, 68 °C for 2 min; and finally 68 °C for 7 min. Aliquots of the RT-PCR products were analyzed on 1% agarose gel by electrophoresis and were subjected to Southern analysis using probes (Table I) for the peptide sequence other than those used in RT-PCR. The RT-PCR fragments were then re-amplified with Taq polymerase (Stratagene) and subcloned into the pT7Blue T-Vector (Novagen).

cDNA Library Screening-- The cDNA fragment amplified with the AP-5 sense primer and the DN-9 antisense primer (Fig. 1, Probe 1) was used to screen the bovine small intestine cDNA library (CLONTECH). The probe was 32P-labeled using a PCR radioactive labeling system (Life Technologies, Inc.). A fragment of approximately 150 bp of the 5'-end of phage clone #1e (Fig. 1, Probe 2) was amplified for labeling and used for the second screening. A similar procedure was carried out using the phage clone #2a sequence (Fig. 1, Probe 3) for the third screening. The insert fragment of each phage was amplified with Pfu polymerase (Stratagene) or Taq polymerase and subcloned into the pCR-Script Amp SK(+) vector (Stratagene) or pT7Blue T-Vector for nucleotide sequencing. Dye Primer and Dye Terminator cycle sequencing kits (Perkin-Elmer) and an ABI sequencer model 377 (Perkin-Elmer) were used for DNA sequencing.

5'-Rapid Amplification of cDNA Ends (RACE)-- 5'-RACE was carried out by a modification of a published method (27). Bovine small intestine poly(A)+ RNA was reverse-transcribed with RRT1 primer (5'-TATTGAAACTCCTGTTCGTCCATTTCCGAT-3'), and a poly(dA) tail was added with terminal deoxynucleotidyltransferase (Stratagene). The products were subjected to PCR using Pfu polymerase and three primers: RPCR1 (5'-CTGCACGGCAGGTTGAAGGCTTCCT-3') as the antisense primer (50 pmol); adaptor (50 pmol), 5'-GCCGCATGCGAATTCACC-3'; and adaptor-(dT)17 (6 pmol). PCR was set at 94 °C for 5 min followed by 20 cycles of 94 °C for 30 s, 50 °C for 1 min, 72 °C for 1 min; 20 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min; and finally 72 °C for 5 min. The PCR products were purified twice with Centricon 100 (Amicon) to remove any excess primers and were then cloned into the pCR-Script Amp SK(+) vector. This library was screened with Probe 3 (Fig. 1) by colony hybridization. The second 5'-RACE was performed based on the DNA sequence of the clone resulting from the first 5'-RACE. Primer sequences were RRT2 (5'-AATTGCTGTACAATGGCACTGAGCTCAGA-3') for reverse transcription and RPCR2 (5'-AGAGCGCTGAGAGATTCGA-3') for PCR. The library of the second 5'-RACE clones was screened with Probe 4 (Fig. 1).

Plasmid Constructions-- An Eco47III-HpaI fragment of the 5'-RACE clone #3a, a HpaI-BstXI fragment of phage clone #2a, and a BstXI-KpnI (in vector) fragment of #1a phage insert cloned in the pT7Blue T-Vector were sequentially ligated between the Eco47III and KpnI (in vector) sites of the 5'-RACE clone #4a in the pCR-Script Amp SK(+) vector. The resultant plasmid, pCRBGT4, was used as a template for the following PCR experiments to make the various expression vectors (Fig. 1).

Transient Expression and GnT-IV Assay-- The entire open reading frame of bovine GnT-IV was amplified with primers 4EXPF2 (introducing an XhoI site upstream of the ATG codon) and 4EXPR (introducing an XbaI site downstream of the stop codon), digested with XhoI and XbaI, and cloned into the pSVL vector (Amersham Pharmacia Biotech) to produce the expression vector pBGT4. Expression vectors with serial N-terminal deletions ligated to the EPO signal sequence (28) were constructed by overlapping PCR as follows: a cDNA fragment encoding the EPO signal sequence was amplified with the XhoEsig primer (introducing an XhoI site upstream of the ATG codon of the EPO signal sequence) and an antisense primer encoding junction sequence 1 (Table I). On the other hand, the GnT-IV segment was amplified with a sense primer for junction sequence 1 and the 4EXPR primer. Aliquots of both PCR products were combined and amplified again with the primers XhoEsig and 4EXPR. The final PCR product was digested with XhoI and XbaI and cloned into pSVL, resulting in pSigIle93. Similar procedures were employed for junction sequences 2 and 3, resulting in pSigPro113 and pSigPro142, respectively. Serial C-terminal deletion was carried out by PCR using the 4EXPF2 primer in combination with a primer with a TGA codon and an XbaI restriction site following the GnT-IV coding region terminating at Gly-499, Pro-465, Lys-432, or Pro-383. The amplified fragments were subcloned into pSVL, giving rise to expression plasmids pCGly499, pCPro465, pCLys432, and pCPro383, respectively. Codons for Ser-79 and Thr-460 were changed into Ala codons by PCR (AGT to GCT and ACG to GCG, respectively) to abolish potential N-glycosylation sites at Asn-77 and Asn-458, respectively. All constructs (Fig. 4) used for transient expression were confirmed by DNA sequencing.

Determination of GnT-IV Activity-- The GnT-IV assay was performed according to the method of Oguri et al. (24), which is a modification of the method of Nishikawa et al. (29). The substrate Gn2(2',2)core-PA was prepared as reported previously (30). 10 µg of each expression plasmid was incorporated into 5 × 106 of COS-7 cells in 0.8 ml of phosphate-buffered saline by electroporation using a Gene Pulser (Bio-Rad) and conditions of 1,600 V, 25 µF at room temperature. After 72 h of cultivation in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.) at 37 °C, the cells were collected, suspended in 100 µl of buffer (5 mM Tris-Cl, pH 7.5, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride), and disrupted by sonication. The supernatants were collected by centrifugation at 2,000 × g for 5 min and used to measure GnT-IV activity. The GnT-IV activity of each lysate was normalized to its protein concentration to reflect the cell number.

Detection of mRNA Level by RT-PCR-- RT-PCR was performed with poly(A)+ RNA from various bovine tissues and the b1NPF (5'-CCATAGTGGCAACCAGGATCATC-3') and b1NPR (5'-CGAAATGGGGTTTAGGCTGG-3') primers. 25 pmol of each primer was used in a 25-µl reaction using an Access RT-PCR kit. The mRNA level was reduced until the difference in the amplified fragments among the various tissues became obvious. The temperature cycle employed was 48 °C for 45 min, 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 57 °C for 1 min, 68 °C for 2 min; and finally 68 °C for 7 min. Glyceraldehyde 3-phosphate dehydrogenase mRNA was amplified with primers, 5'-CCAAAATCAAGTGGGGCGATG-3' and 5'-CAGGAGGCATTGCTGATGATCTTG-3' as a control. Poly(A)+ RNA from bovine small intestine was purified as described above. Poly(A)+ RNAs from other tissues were purchased from CLONTECH.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Microsequencing of Purified GnT-IV-- Using 300 pmol of the purified GnT-IV preparation, we obtained 31 peptide fractions (16 from Achromobacter protease I digestion and 15 from the following Asp-N treatment). Twenty of the fragments (7 from the former and 13 from the latter treatment) were subjected to peptide sequencing, and 24 peptide sequences were obtained, since some fractions gave mixed signals. Four peptide sequences were identified as being fragments derived from the bovine IgG heavy chain, whereas the others were novel. We also determined the amino acid sequence of the N terminus of GnT-IV prepared free of IgG fragments as NH2-Ile-Leu-Lys-Glu-.

cDNA Library Screening-- We performed RT-PCR using bovine small intestine poly(A)+ RNA as a template and primers corresponding to four novel amino acid sequences, as shown in Table I and Fig. 2. A 0.2-kilobase fragment amplified with the AP-5 sense primer and the DN-9 antisense primer (Table I) was cloned and sequenced. The deduced amino acid sequence encoded by this cDNA included four other peptide fragments that were not employed in the RT-PCR strategy, indicating that this amplified fragment is part of the GnT-IV cDNA and could be used for screening of the cDNA library (Fig. 1, Probe 1).

                              
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Table I
Oligonucleotides used for RT-PCR and construction of the expressed fusion proteins
The C termini for erythropoietin signal sequences are shown by left-down arrow  and the peptide region of GnT-IV in each construct is underlined. Junction sequences 1, 2, and 3 were designed to fuse the erythropoietin signal sequence with the GnT-IV coding sequence starting from Ile-93, Pro-113, and Pro-142, respectively.


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Fig. 1.   Restriction map and regions of clones on bovine cDNA fragment of GnT-IV. Restriction map of cDNA fragment is shown at the top. The open bar indicates the open reading frame of GnT-IV and the shaded area is the putative transmembrane region. phi , phage clone; R, 5'-RACE clone. The hatched boxes show the probe regions spanning 1,446-1,621 (Probe 1), 993-1,142 (Probe 2), 494-643 (Probe 3) and 9-60(gap)382-445 (Probe 4). The thin lines in the RACE clones indicate gaps in the nucleotide sequence (see "Discussion"). Delta  indicates the N terminus of the purified bovine GnT-IV. N in the open bar means a potential N-glycosylation site (Asn-Xaa(except Pro)-Ser/Thr).

The nucleotide sequences of five independent clones (Fig. 1, phi #1a to 1e) from the first screening showed that they shared the 3'-terminal portion of GnT-IV coding region (Fig. 1). We could not obtain the initiation codon by the second and third screenings using the 5'-terminal region of the #1e and #2a phage inserts (Fig. 1, Probe 2 and 3, respectively). Thus, we performed 5'-RACE to obtain the 5'-portion of the open reading frame. After two cycles of experiments, the complete open reading frame was obtained (Figs. 1 and 2).


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Fig. 2.   Nucleotide and deduced amino acid sequences of GnT-IV cDNA. A bovine cDNA fragment of 2,246 bp was sequenced. The underlined areas indicate amino acid sequences matched with the peptide fragments of the purified bovine GnT-IV, and those sequences with names (AP-15, AP-12, AP-5, and DN-9) were used for primer design. Asn at 458 did not produce any signal in peptide sequencing (data not shown). The double underlined amino acids form the putative transmembrane domain. # and Delta  indicate a potential N-glycosylation site and the N terminus of the purified GnT-IV, respectively.

Nucleotide and Amino Acid Sequences of GnT-IV-- The 2,246-bp cDNA fragment obtained (Fig. 2) consisted of a 1,605-bp open reading frame, 287 bp upstream and 354 bp downstream regions. The first methionine codon at 288-290 (Fig. 2) is considered to be the only initiation site, since no other in frame methionine codon was found, and the nucleotide sequence around 288-290 is partially consistent with Kozak's rule (31). Neither a poly(A) tail nor the AATAAA polyadenylation initiation signal was found within the 3'-untranslated region obtained. The polypeptide deduced from this open reading frame consists of 535 amino acids (Fig. 2) and is expected to be 61,613 Da. 19 of the novel peptide sequences obtained from the purified bovine GnT-IV appeared in this open reading frame (Fig. 2). N-terminal sequence analysis of the purified GnT-IV revealed that most of the enzyme was truncated at Ile-93, indicating proteolysis during enzyme purification; heterogeneity of the end was observed. Hydrophobicity and the surface probability profile showed that an 18-amino acid (position 8-25) region close to the N terminus could be a transmembrane domain, suggesting that GnT-IV is a typical type II membrane protein (Fig. 3). Potential N-glycosylation sites (Asn-Xaa(except Pro)-Ser/Thr) were found at Asn-5, -77, and -458, whereas O-glycosylation motif preferred by bovine O-GalNAcT1 (Thr/Ser-Pro-Xaa-Pro or Thr-Xaa-Xaa-Pro) (32) was not found.


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Fig. 3.   Hydrophobicity profile of GnT-IV. Hydrophobicity was calculated by the algorithm of Kyte and Doolittle (48). The closed and open bars indicate the putative transmembrane domain and the putative stem region, respectively. The vertical and horizontal axes show hydrophobicity and the position of amino acids deduced from the GnT-IV cDNA.

Expression of GnT-IV Activity-- The entire open reading frame in the pSVL vector was transiently expressed in COS-7 cells. The transfected cells showed a >50-fold increase in in vitro GnT-IV activity (Fig. 4, pBGT4). N-terminal deletion up to Ile-93 (pSigIle93), which is identical to the purified bovine GnT-IV, still expressed GnT-IV activity and the truncated enzyme could be secreted using the EPO signal sequence. On the other hand, deletions up to Pro-113 (pSigPro113) and Pro-142 (pSigPro142) abolished GnT-IV activity.


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Fig. 4.   Peptide regions of GnT-IV mutants and their activity when transiently expressed in COS-7 cell. The open bar at the top shows the open reading frame of GnT-IV with potential N-glycosylation sites (N), the transmembrane domain (shaded area), and the N terminus of the purified enzyme from bovine small intestine (Delta ). The solid bar indicates the region cloned in each plasmid. The hatched bar shows the EPO signal sequence. X means that N-glycosylation was abolished by amino acid substitution. The GnT-IV activity of each construct is shown on the right as percentage against that obtained with pBGT4. The GnT-IV activity secreted into the medium is also indicated as a percentage as above when applicable (in parentheses). All results are the averages at least three experiments.

We also have expressed serial deletion mutants at the C terminus of GnT-IV in COS-7 cells. Contrary to our expectation, pCPro383, which had the largest deletion of all the C-terminal deletion mutants, maintained complete GnT-IV activity in vitro (Fig. 4). Although we did not make further deletions in the C-terminal portion, from these two deletion experiments, we concluded that the catalytic domain of bovine GnT-IV must be located between Ile-93 and Pro-383.

Amino acid substitutions of Ser-79 and Thr-460 with Ala, which disrupted the consensus sequence of N-glycosylation at Asn-77 and Asn-458, respectively, did not significantly reduce GnT-IV activity (Fig. 4, pS79A and pT460A).

Comparison of GnT-IV Expression in a Variety of Bovine Tissues-- We performed RT-PCR using poly(A)+ RNA to compare GnT-IV mRNA abundance between various bovine tissues (Fig. 5). The amount of amplified bands differed between tissues. Small intestine, kidney, lung, and spleen gave strong signals for GnT-IV mRNA, whereas brain, heart, and liver produced faint signals.


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Fig. 5.   RT-PCR analysis of GnT-IV mRNA. Messenger RNAs from various bovine tissues were subjected to RT-PCR using a GnT-IV cDNA specific primer set (top panel) or a glyceraldehyde 3-phosphate dehydrogenase cDNA specific primer set (bottom panel). The detailed sequences of the primers are described under "Experimental Procedures."

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have isolated a cDNA fragment containing the entire open reading frame for bovine GnT-IV based on the amino acid sequences of the purified bovine enzyme. The open reading frame of 535 amino acids within the cDNA fragment obtained was confirmed to encode GnT-IV by two methods: 1) it contained 19 of the 20 novel peptide sequences of the bovine GnT-IV preparation (Fig. 2); 2) it conveyed GnT-IV activity in vitro by transient expression in COS-7 cells (Fig. 4).

Comparison of the amino acid sequence of bovine GnT-IV with those of other GnTs revealed no obvious homology, whereas all GnTs including GnT-IV shared a type II transmembrane protein structure. The region between the end of the transmembrane domain of GnT-IV and Ile-93 is rather hydrophilic (Fig. 3) and flexible, suggesting that this region could be the stem region. In addition, the protease lability of this sequence also suggested that this region was exposed. Although many other glycosyltransferases carry proline-rich sequences in the stem region (10, 17, 33), GnT-IV does not.

The purified GnT-IV had N-glycan(s), because it was sensitive to N-glycosidase digestion (24). There are three potential N-glycosylation sites, Asn-5, Asn-77, and Asn-458; however, the purified enzyme had lost Asn-5 and Asn-77. Therefore, Asn-458 is the only N-glycosylation site in the truncated GnT-IV. Indeed, no amino acid could be detected at the Asn-458 position in peptide sequencing, indicating that this residue might be modified (data not shown). Taking all evidence together, it was concluded that the purified GnT-IV had one Asn-linked oligosaccharide at Asn-458, although this sugar chain was not essential for GnT-IV activity (Fig. 4). It is possible that Asn-5 is not N-glycosylated, because it is presumably located on the cytosol side. There is no evidence to suggest the glycosylation state of Asn-77.

During purification of GnT-IV from bovine small intestine, we added protease inhibitors in the complete procedure to avoid proteolysis. Nevertheless, the GnT-IV thus obtained appeared to be truncated at Ile-93. The calculated molecular weight of the polypeptide extending from Ile-93 was 51,000. The difference in molecular weight between the deduced peptide above and the estimated size of the purified enzyme obtained by SDS-polyacrylamide gel electrophoresis (58,000; Ref. 24) was much larger than the molecular weight of one Asn-linked oligosaccharide. This might indicate a maturation procedure other than N-glycosylation. Limited proteolysis during purification has also been observed for other glycosyltransferases (10, 14, 17, 20, 34), which suggests that truncations of glycosyltransferases occur naturally and have in vivo functions.

N- and C-terminal deletion experiments indicated that the region bearing in vitro GnT-IV activity was between Ile-93 and Pro-383. This region had many lysines that might be involved in UDP binding (35, 36). So far, there is little data regarding the tertiary structures of GnTs, and the substrate binding site has not been specified. Although all GnTs share the common donor substrate, UDP-GlcNAc, there is no consensus motif in their amino acid sequences. Indeed, the GnTs purified so far have quite different Km values for UDP-GlcNAc (0.04 mM for GnT-I, 0.96 mM for GnT-II, 3.1 mM for GnT-III, 0.22 mM for GnT-IV2, and 11 mM for GnT-V) (20, 37-39). Therefore, it is possible that each enzyme has a different means of binding the donor substrate. We would like to elucidate the structure and function of GnT-IV and compare this data with other GnTs when available.

Our attempt to produce soluble forms of GnT-IV showed that the replacement of the transmembrane and stem regions with the EPO signal sequence was not enough for full secretion (Fig. 4, pSigIle93). There might be a signal located between Ile-93 and the C terminus of GnT-IV to retain the protein within the cell, although such signals are usually considered to lie within the transmembrane domain and nearby regions (40-43). Currently, we are attempting to raise antibody against GnT-IV, which would help to determine the localization of GnT-IV in recombinant cells, as well as naturally occurring cells and tissues.

Partial information regarding the intron/exon organization was obtained from the 5'-RACE experiments. A total of three 5'-RACE clones was obtained containing different cDNA fragments. The 5'-RACE clones #3a and #4b had gaps in their cDNA from residues 61 to 381 and 61 to 185, respectively (Figs. 1 and 2). On the other hand clone #4a had a continuous segment starting from residue 34. Complementary DNA sequences preceding the ends of the gaps in clones #3a and #4b were both similar to the consensus sequence of the intron acceptors (44). Comparison between a bovine partial genomic clone and the cDNA segments obtained revealed that there were at least three introns in the GnT-IV genomic DNA corresponding to 1-550 bp of the cDNA sequence and the splicing variation had occurred in this region (data not shown). GnT-I and -II are encoded as single exons, whereas GnT-III and -V have complex genomic organizations (11-13, 16, 19, 23). GnT-IV could have many exons/introns, and this might be a common feature of genomic DNAs encoding branch-forming enzymes for N-glycans.

We analyzed the expression level of GnT-IV in various bovine tissues by RT-PCR. The result showed different levels of GnT-IV mRNA in these tissues, indicating that GnT-IV expression is regulated. The small intestine, which was used as a source of mRNA for cloning as well as being the source of enzyme for purification, produced a strong GnT-IV mRNA signal. The spleen also gave a strong signal, which was coincident with its high GnT-IV activity,2 whereas the heart, which had an undetectable level of activity,2 produced a weak signal. The tissue specific expression pattern of GnT-IV may suggest the importance and role of the beta 1-4 GlcNAc branch in certain cellular functions. The branching structure of N-glycans could be, in part, controlled by the relative activities of the various enzymes involved in branch formation, such as GnT-III, -IV, -V, and beta 1-4 galactosyltransferase. A bisecting GlcNAc produced by GnT-III and a galactose residue attached to either of two beta 1-2 GlcNAcs inhibited GnT-IV action, whereas the beta 1-6 GlcNAc at the alpha 1-6 mannose, which is a product of GnT-V, enhanced GnT-IV activity (24). It has been suggested that the increase of the antenna number of Asn-linked sugar chains in certain cell lines and tumor tissues is mainly due to the elevation of GnT-V activity (45-47). Since GnT-IV activity is also essential to produce tetra-antennary structure, its increase together with GnT-V could contribute to cellular changes that require GnT-IV product specifically.

There was no method for the detection of the GnT-IV enzyme immunologically or its product of the beta 1-4 GlcNAc branch at the alpha 1-3 Man. This could be one reason that the role of GnT-IV in a variety of phenomena is not well understood. Using the GnT-IV cDNA reported here, it became possible to evaluate the expression level of GnT-IV. Using the cDNAs of all the GnTs and beta 1-4 galactosyltransferase involved in N-glycan branch formation it will be possible to compare the expression levels of the GnTs to clarify the control of branching. Overexpression and/or suppression of these enzymes will provide tools for producing glycoproteins with targeted antennary sugar chain structures. Also, an enzymatic approach will now be available to elucidate the factors that determine the branch formation of N-glycans using the recombinant GnT-I-V enzymes. Moreover, these recombinant enzymes will be very useful to remodel the branch structure of N-glycans attached to glycoproteins to see if they alter the biological properties of the proteins.

    ACKNOWLEDGEMENTS

We are very grateful to Prof. Naoyuki Taniguchi (Osaka Univ.) for helpful discussions. We also thank Dr. Shinji Takamatsu (KIRIN Brewery) for critical reading of this manuscript.

    FOOTNOTES

* This work was supported by New Energy and Industrial Technology Development Organization (NEDO) as a part of the Research and Development Projects of Industrial Science and Technology Frontier Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000628.

Dagger Research fellow of New Energy and Industrial Technology Development Organization (NEDO).

§ Present address: Dept. of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan.

To whom correspondence should be addressed: Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., 1-13-5 Fuku-ura, Kanazawa-ku, Yokohama, 236-0004, Japan. Tel.: 81-45-788-7200; Fax: 81-45-788-4047.

1 The abbreviations used are: GnT-IV, UDP-N-acetylglucosamine:alpha 1,3-D-mannoside beta 1,4-N-acetylglucosaminyl transferase; GnT, N-acetylglucosaminyltransferase; GlcNAc, N-acetyl-D-glucosamine; Man, mannose; GnT-V, UDP-N-acetylglucosamine:alpha 1,6-D-mannoside beta 1,6-N-acetylglucosaminyltransferase V; EPO, erythropoietin; GnT-I, UDP-N-acetylglucosamine:alpha 1,3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase; GnT-II, UDP-N-acetylglucosamine:alpha 1,6-D-mannoside beta 1,2-N-acetylglucosaminyltransferase; GnT-III, UDP-N-acetylglucosamine:beta -D-mannoside beta 1,4-N-acetylglucosaminyltransferase; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; bp, base pair(s); Gn2(2',2)core-PA, GlcNAcbeta 1-2Manalpha 1-6(GlcNAcbeta 1-2Manalpha 1-3)Manbeta 1-4GlcNAcbeta 1-4GlcNAc-pyridylamine; RACE, rapid amplification of cDNA ends.

2 S. Oguri, M. T. Minowa, Y. Ihara, N. Taniguchi, H. Ikenaga, and M. Takeuchi, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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