Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine:[alpha]1,3-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase

Aruto Yoshida, Mari T. Minowa, Shinji Takamatsu, Tomoka Hara, Suguru Oguri2, Hiroshi Ikenaga and Makoto Takeuchi1

Glycotechnology Group, Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan

Received on June 16, 1998; revised on July 17, 1998; accepted on July 21, 1998

A human cDNA for UDP-N-acetylglucosamine:[alpha]1,3-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.

Key words: N-acetylglucosaminyltransferase/N-glycosylation/cDNA cloning/expression/chromosomal mapping

Introduction

Branching structures in complex N-glycans are synthesized in the Golgi apparatus by N-acetylglucosaminyltransferase (GnT) -I to -VI on a common core structure of Man[alpha]1-6(Man[alpha]1-3)Man[beta]1-4 GlcNAc[beta]1-4GlcNAc[beta]1-Asn (Schachter et al., 1989). UDP-N-acetylglucosamine:[alpha]1,3-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase (GnT-IV; EC 2.4.1.145) catalyzes the transfer of GlcNAc from UDP-GlcNAc in [beta]1-4 linkage to [alpha]1,3-d-mannoside on GlcNAc[beta]1-2Man[alpha]1-6(GlcNAc[beta]1-2Man[alpha]1-3)Man[beta]1-4 GlcNAc[beta]1-4GlcNAc[beta]1-Asn, which is the common substrate for GnT-III, -V and [beta]1,4-GalT (Gleeson and Schachter, 1983; Schachter et al., 1989). Therefore, GnT-IV is one of the key glycosyltransferases regulating formation of tri- and other multiantennary structures. Previous reports illustrated that GnT-IV is expressed in avian and many mammalian species (Gleeson and Schachter, 1983; Yamashita et al., 1985; Koenderman et al., 1987; Nakao et al., 1990; Nishikawa et al., 1990; Ogier-Denis et al., 1990). However, GnT-IV activity was generally lower than other branch forming enzyme activities in many tissues and cell lines (Yamashita et al., 1985; Koenderman et al., 1987; Nakao et al., 1990; Nishikawa et al., 1990). Recently, high GnT-IV activity was found in bovine small intestine and the enzyme was purified to homogeneity by Oguri et al. (Oguri et al., 1997) in our laboratory. To date, cDNAs of GnT-I (Kumar et al., 1990, 1992; Sarkar et al., 1991), -II (D'Agostaro et al., 1995; Tan et al., 1995), -III (Nishikawa et al., 1992; Ihara et al., 1993), and -V (Shoreibah et al., 1993; Saito et al., 1994) have been cloned and characterized, and their patterns of expression have been surveyed (Kumar et al., 1992; Miyoshi et al., 1993, 1995; Tan et al., 1995). In contrast, expression of GnT-IV mRNA could not be examined due to the lack of the cDNA.

The highly branched complex type N-glycans on glycoproteins have been reported to be deeply related to several biological phenomena. Takeuchi et al. (Takeuchi et al., 1989) reported in human erythropoietin (hEPO) that the ratio of tetra-antennary to biantennary sugar chains on the protein correlates positively with its activity, indicating that branch number of N-glycans regulates in vivo biological activity. A pharmacokinetic study showed that the low activity of hEPO with biantennary N-glycans is due to its rapid clearance from the blood circulation by the kidney. In contrast, hEPO with well-branched tetra-antennary glycans remained at higher levels in the plasma (Misaizu et al., 1995). Hepatocyte asialoglycoprotein receptor binds with the highest affinity to three galactose residues on a triantennary structure of asialoglycoproteins (Rice et al., 1990). The receptor consists of a major and a minor subunits, and the latter subunit rigidly recognizes a galactose located on GlcNAc[beta]1-4Man[alpha]1-3 branch. GnT-IV catalyzes formation of this branch and thus its activity may also regulate the clearance of aged or activated glycoproteins in the blood circulation.

The regulation of GnT-IV expression is particularly interesting because enzymatic activity increases during oncogenesis and differentiation. Nishikawa et al. (Nishikawa et al., 1990) showed that a solid ascites hepatoma cell line, AH-130, has higher GnT-IV activity than normal liver tissue. The structural analysis of N-glycans on [gamma]-glutamyltranspeptidase from human hepatoma tissue also suggests that hepatocarcinogenesis increases GnT-IV activity (Yamashita et al., 1989). A similar increase in GnT-IV activity may occur in choriocarcinogenesis because aberrant triantennary sugar chains containing GlcNAc[beta]1-4Man[alpha]1-3 branch appear in human chorionic gonadotropin from patients with choriocaricinoma (Mizuochi et al., 1983). Upregulation of GnT-IV activity has also been reported during differentiation of human myelocytic cells by 1[alpha],25-dihydroxyvitamine D3 (Koenderman et al., 1987) and interleukin-6 (IL-6) (Nakao et al., 1990). These observations imply that GnT-IV has a biological role in regulating the availability of serum glycoproteins, oncogenesis, and differentiation.

To examine how GnT-IV expression is regulated at molecular level, we isolated a human GnT-IV cDNA. The cDNA encoded a protein of 535 amino acids whose sequence is highly homologous to the bovine counterpart (Minowa et al., 1998). High levels of GnT-IV mRNA existed in tissues containing T lineage cells. Furthermore, the cDNA was used to map the GnT-IV gene to chromosome 2, band q12. Isolation of this cDNA permits future investigations regarding the role of human GnT-IV in modulating branch formation in N-glycans.

The sequence reported in this article has been deposited in the DDBJ/GenBank/EMBL data base (accession no. AB000616).

Results

Isolation and characterization of human GnT-IV cDNA

To isolate a human homologue to bovine GnT-IV cDNA (Minowa et al., 1998), primers derived from bovine GnT-IV (1-2F and 1-1R in Figure 1) were used in RT-PCR containing human liver RNA as a template. A 0.65 kb fragment was specifically amplified and sequence analysis revealed that this fragment contained a short reading frame, which was highly homologous to bovine GnT-IV cDNA (Minowa et al., 1998). This fragment was then used to screen a human liver cDNA library. Two independent phage clones, hGT4-1/[lambda]1 and hGT4-1/[lambda]2, were isolated after screening approximately 1.6 × 106 recombinant phages. Restriction digests and nucleotide sequence analysis of these clones revealed that hGT4-1/[lambda]1 was completely included in hGT4-1/[lambda]2 (Figure 1). Clone hGT4-1/[lambda]2 was ~2.1 kb and contained an open reading frame (ORF) of 1605 bp (Figure 2). This ORF was the same in size to that of the bovine GnT-IV (Minowa et al., 1998). The putative initiation site is the ATG at position 136-138, since it is downstream of five stop codons in the 5[prime] untranslated regions of 135 bp. A termination codon appeared at position 1741-1743, however, the polyadenylation consensus sequence of AATAAA was not found in the partial 3[prime] untranslated region. The ORF encodes a 535 amino acid protein with a calculated molecular mass of 61.5 k. Hydrophobicity analysis (Kyte and Doolittle, 1982) and secondary structure prediction (Chou and Fasman, 1974) of the amino acid sequence indicated amino acids at region 8-24 are capable of folding into an [alpha]-helix and forming a putative transmembrane domain. This prediction suggests that human GnT-IV is a type II membrane protein similar to other Golgi glycosyltransferases cloned to date (Paulson and Colley, 1989; Kleene and Berger, 1993; Taniguchi and Ihara, 1995). There are three potential N-glycosylation sites at Asn5, Asn77, and Asn458 having the consensus sequence of Asn-Xaa-Ser/Thr (Xaa[ne]Pro) (Ronin et al., 1978).


Figure 1. Restriction map of human GnT-IV cDNA clones. The protein coding region and the 5[prime]- and 3[prime]-noncoding regions of GnT-IV cDNA are represented by an open box and thin lines, respectively. An arrow in the box indicates the direction of transcription. Shadowed bars, thick bars, and an open bar indicate the probe regions, the inserts of phage clones and the expressed region, respectively.


Figure 2. Nucleotide and amino acid sequences of human GnT-IV. The deduced amino acid sequence of GnT-IV is given below the nucleotide sequence. The putative transmembrane domain is underlined. Asterisks under asparagine residues indicate potential N-glycosylation sites (Asn-X-Ser/Thr) (Ronin et al., 1978).

The nucleotide and predicted amino acid sequences of human GnT-IV were compared with those of the bovine enzyme (Minowa et al., 1998). The nucleotide sequences were 91% homologous in the coding region and more than 80% homologous in both partial 5[prime] and 3[prime] noncoding regions. The predicted amino acid sequence were 96% identical, indicating that GnT-IV is highly conserved between species. All three potential N-glycosylation sites and four cystein residues were detected at the same positions. It has been reported that interspecies sequence heterogeneity of several other glycosyltransferases primarily occurs in the cytoplasmic tails and/or the stem regions (Kleene and Berger, 1993). Similarly, 8 of 21 differences between bovine and human GnT-IV were observed in the short stem domain. The remaining 13 differences were randomly scattered throughout the remainder of GnT-IV.

Transient expression of human GnT-IV cDNA in COS7 cells

The putative coding region was subcloned into the mammalian expression vector pSVL, and the resulting plasmid pHGT4-1 was transfected to COS7 cells. After 3 days culture, lysates of these transfectants were assayed for GnT-IV activity using Gn2(2',2)core-PA as an acceptor. Cells transfected with the human cDNA exhibited ~25-fold higher GnT-IV activity than cells transfected with the null vector (Table I), demonstrating that this cDNA sequence encodes a functional GnT-IV. Based on the previous report of transfection efficiency in this system (Yoshida et al., 1995), it was estimated that the human cDNA increased GnT-IV activity about 500-fold per transfected cell.

Table I. GnT-IV activity in COS7 cells transiently expressing human liver GnT-IV cDNA
Plasmid GnT-IV activity (pmol/h/mg protein) -fold
pSVL 1037 1.0
pHGT4-1 25,370 ± 3582 24.5
pBGT4a 33,402 32.2
GnT-IV activities of cell lysates were measured by using Gn2(2',2)core-PA as an acceptor. The mean and deviation were determined by two independent transfectants.
aThe plasmid expressing bovine GnT-IV is described in Minowa et al. (1998).

Northern blot analysis of human GnT-IV in human tissues and cell lines

Using probe 2 and a G3PDH probe (as a control), the expression of human GnT-IV mRNA in twenty three human normal tissues and eight cancer cell lines was analyzed by Northern hybridization (Figure 3). Although signals for several tissues are almost invisible in Figure 3, all tissues and cell lines exhibited five bands of different sizes in the same blot after longer exposure; three bands (approximate mRNA sizes of 9.7, 3.8, and 2.4 kb) were the most intense whereas the remaining two bands (7.6 and 5.1 kb) were less intense. The relative expression levels of these five bands to one another were similar in different tissues and cell lines. An identical pattern of bands was observed when these membranes were hybridized with probe 3 in Figure 1 further indicating that each band contains GnT-IV sequence (data not shown). Although an additional band of 1.8 kb was present for peripheral blood leukocyte in Figure 3, this result could not be reproduced in other blots (data not shown). The expression levels of GnT-IV mRNA were significantly different among tissues and cell lines. Of the normal tissues examined, pancreas, spleen, thymus, prostate, small intestine, peripheral blood leukocyte, and lymph node expressed high levels of GnT-IV mRNA. Of the cell line tested, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 exhibited the strongest signals. Although the colorectal adenocarcinoma cell line SW480 showed high expression in Figure 3, its appearance could not be confirmed by another blot (data not shown). Not all cancer cell lines express a significant amount of GnT-IV mRNA, while previous studies suggested the upregulation of GnT-IV expression by several malignant alterations (Mizuochi et al., 1983; Yamashita et al., 1989; Nishikawa et al., 1990). Further investigations are required to understand the influence of each oncogenesis on GnT-IV expression.


Figure 3. Northern analysis of GnT-IV expression in human tissues and cancer cell lines. Human multiple tissue Northern blots (Human MTN, Human MTN II, Human MTN III, and Human cancer cell line MTN) containing ~2 µg of poly(A)+ mRNA per lane were hybridized to the 32P-labeled Probe 2 shown in Figure 1 (upper panel). The blots were re-hybridized to the 32P-labeled human G3PDH cDNA probe as a control (lower panel). The sizes of RNA marker bands are indicated on the left.

Chromosomal localization of human GnT-IV gene

The chromosomal location of human GnT-IV gene was determined by fluorescence in situ hybridization analysis. Using the primers 1-9F and 1-8R, PCR produced a 486 bp genomic DNA which contained an intron of 130 bp. A human P1 library was screened by PCR using the same primers, and a P1 plasmid clone containing human GnT-IV gene, F235, was isolated. This clone was then used as a probe for in situ hybridization. In the initial experiment, this probe hybridized to the proximal long arm of a group A chromosome. The following cohybridization using a probe from N-myc locus as a marker (specific for the chromosome 2 band p23-24; Schwab et al., 1984) revealed that clone F235 exists in the long arm of the same chromosome. Measurements of 10 specifically hybridized chromosomes 2 demonstrated that GnT-IV is located at a position which is 9% of total distance from the centromere to the telomere of chromosome arm 2q. This position corresponds to 2q12 (Figure 4). Signals were observed in 75% (60 out of 80) of the cells examined, and most of them were detected on both chromatids of a single chromosome.


Figure 4. Mapping of the human GnT-IV gene by in situ hybridization. A solid arrowhead schematically represents the location of human GnT-IV gene. An open arrowhead indicates the location of human GnT-V gene reported by Saito et al. (Saito et al., 1994).

Discussion

It has been observed that the number of antennae in complex type N-glycans of mammalian cells are modulated during oncogenesis (Mizuochi et al., 1983; Yamashita et al., 1989) and development (Berjonneau et al., 1984; Codogno et al., 1985). Furthermore, an increase of GnT-IV activity has been indicated in myelocytic differentiation (Koenderman et al., 1987; Nakao et al., 1990), hepatocarcinogenesis (Nishikawa et al., 1990), and embryo development (Ogier-Denis et al., 1990). Nonetheless, these modulations are quite complex processes because several glycosyltransferases (e.g., GnT-III, -IV, -V, and [beta]1,4-GalT) compete for the same biantennary oligosaccharide as a substrate. Therefore, it is necessary to analyze the expression and activity of these glycosyltransferases concomitantly. Human cDNAs encoding GnT-III (Ihara et al., 1993), -V (Saito et al., 1994), and [beta]1,4-Gal T (Masri et al., 1988) have been identified and characterized. In the present study, we compliment these investigations by isolating the human GnT-IV cDNA. This will facilitate future examination on the regulation of GlcNAc branch formation in complex type N-glycans, as well as its biological role in humans.

Northern analysis indicates that human GnT-IV mRNA exists in multiple sizes, suggesting that the enzyme has more than one site of transcriptional initiation, alternative splicing, or a combination of these processes. Expression levels of human GnT-IV mRNA varied between normal tissues and between cell lines (Figure 3). This result demonstrates tissue specific expression of the GnT-IV mRNA. Our data illustrating GnT-IV mRNA expression in human tissues generally agrees with data on rat GnT-IV activity in corresponding tissues (Nishikawa et al., 1990). Moreover, GnT-IV activity of several cell lines roughly correlated with their amount of GnT-IV mRNA in Northern analysis (Table II, Figure 3). The GnT-IV activity assay using oligosaccharide substrate also showed that HL-60 and MOLT-4 express larger amount of GnT-IV than HeLa S3, K-562, Raji, and A549 (Table II).

Table II. GnT-IV activity in various cell lines
Cell line GnT-IV activity (pmol/h/mg protein)
HL-60 557.8
HeLa S3 80.7
K-562 103.0
MOLT-4 1133.5
RAJI 29.8
A549 75.7
GnT-IV activities of various cell lines were measured by the method described in Materials and methods. Cell lines are obtained from Japanese Collection of Research Bioresources (for HL-60, K-562, and RAJI) or RIKEN Cell Bank (for HeLa S3, MOLT-4, and A549). Each cell line was cultured in the medium specified by the providers.

The present investigation demonstrates that lymphoid tissues such as spleen, thymus, small intestine, lymph node, and peripheral blood leukocyte express a large amount of the GnT-IV mRNA. Thymus exhibits high levels of the mRNA, whereas bone marrow expresses very little (Figure 3). In addition, the T lineage cell line MOLT-4 expresses higher levels of the GnT-IV mRNA and activity than those of the B lineage cell line Raji (Figure 3, Table II). These results suggest that GnT-IV is expressed at a higher level in T lymphocytes, and may be important for development of T lymphocytes in the thymus. It has also been indicated that the triantennary oligosaccharide synthesized by GnT-IV is expressed on mouse thymocytes (Yoshida et al., 1991). Moreover, the amount of GnT-IV product in complex type N-glycans on mature mouse T cells increases upon activation by concanavalin A (Yoshida et al., 1991). While the [beta]1-6 GlcNAc branch synthesized by GnT-V has been shown to increase upon activation of human CD4+ and CD8+ T lymphocytes (Lemaire et al., 1994), how the GnT-IV activity is regulated during T lymphocyte activation and differentiation remains unknown.

The myeloid cell line HL-60 shows high expression of GnT-IV in contrast to myeloblastic lymphoma cell line K-562 (Figure 3, Table II). Abundant GnT-IV mRNA in HL-60 is consistent with the high expression of tri- and tetra-antennary complex N-glycans on this cell line (Mizoguchi et al., 1984). Koenderman et al. (Koenderman et al., 1987) reported that the GnT-IV activity is high in macrophages but not in granulocytes and increases during monocytic differentiation, which suggests that GnT-IV may also be important for differentiation of these cells.

High expression of GnT-IV in the leukocytes may also regulate homing of hematopoietic cells to specific tissues. It has been reported that E-selectin, which is an adhesion molecule mediating homing of leukocytes to inflamed tissues, preferentially binds 3-sialyl di-Lewis X structure coupled to the GlcNAc[beta]1-4Man[alpha]1-3 branch synthesized by GnT-IV (Patel et al., 1994).

Fluorescent in situ hybridization demonstrated that the human GnT-IV gene is located on chromosome 2, band q12. Although the human genes for interleukin-1 receptor (Copeland et al., 1991) and protein tyrosine kinase ZAP-70 (Ku et al., 1994) have been mapped to the same locus, no genetic disease has been identified at this locus. As shown in Figure 4, the human GnT-V gene (2q21) located in close proximity to GnT-IV (Saito et al., 1994). In contrast, human GnT-I maps to chromosome 5 (Kumar et al., 1992; Tan et al., 1995), GnT-II to chromosome 14 (Tan et al., 1995), and GnT-III to chromosome 22 (Ihara et al., 1993). Preliminary analysis of genomic organization showed that the human GnT-IV gene has multiple exons similar to GnT-III (Koyama et al., 1996) and -V (Saito et al., 1995). A short intron of 130 bp occurred between nucleotide 1716 and 1717 of GnT-IV cDNA (Figure 2).

The amino acid sequence identity between human and bovine is 96%, and all three potential N-glycosylation sites and four cystein residues were detected at the same positions. Such a high conservation of GnT-IV amino acid sequence suggests that a highly restricted protein structure is required for recognizing an [alpha]1,3 mannoside and several GlcNAc branches of acceptor complex N-glycans (Oguri et al., 1997). Southern blot analysis using probe 2 suggested that GnT-IV exists in many species including monkey, rat, mouse, dog, rabbit, and chicken as well as human and bovine. On the other hand, we could not detect any homologous genes of human GnT-IV in yeast (data not shown).

The cDNA of human GnT-IV contains an extraordinary high A+T content (62%) in the coding region. Pyrimidine nucleotides are more frequently observed than purine nucleotides in the third codons of GnT-IV; nevertheless, C and G are dominant at that position in most human genes (Zhang and Chou, 1993). The sequence for translational initiation of human GnT-IV does not fully satisfy the Kozak consensus sequence (Kozak, 1987). These features may be related to the relatively low activity of the enzyme in many tissues.

The present article demonstrates that the expression of human GnT-IV is strictly regulated in tissues. In addition to the upregulation of the enzyme in differentiation (Koenderman et al., 1987; Nakao et al., 1990), development (Ogier-Denis et al., 1990), and oncogenesis (Mizuochi et al., 1983; Yamashita et al., 1989; Nishikawa et al., 1990), high expression in leukocytes and T cell associated lymphoid tissues was discovered. This implies that the [beta]1-4 GlcNAc branch synthesized by GnT-IV may be important for the differentiation, trafficking, and/or activation of T lymphocytes.

Materials and methods

Isolation of a human cDNA fragment homologous to bovine GnT-IV cDNA

Based on the partial sequence of bovine GnT-IV cDNA (Minowa et al., 1998), the sense oligonucleotide primer 5[prime]-ACGATTGTGCAACAGTTCAAGCGT-3[prime] (1-2F) and the antisense oligonucleotide primer 5[prime]-GGGAGAACTCCAGGATCATCCAGT-3[prime] (1-1R) were synthesized for RT-PCR. Total RNA from human liver (Clontech) was used as a template and other reagents were supplied from Access RT-PCR system (Promega). Amplification by RT-PCR was carried out for 40 cycles (30 s at 94°C, 1 min at 60°C, and 2 min at 68°C) after reverse transcription for 45 min at 48°C and initial denaturation for 2 min at 94°C on a Gene Amp PCR System 9600 (Perkin Elmer). The amplified cDNA (~0.65 kb) was cloned into pCR-Script Amp SK(+) cloning vector (Stratagene). DNA sequencing was carried out using the Dye Primer Cycle Sequencing FS Ready Reaction Kit (Perkin Elmer) and an ABI PRISM 377 DNA sequencer (Perkin Elmer).

Screening of human liver cDNA library

As shown in Figure 1, the 0.65 kb segment resulting from RT-PCR was labeled with [[alpha]-32P]dCTP (Amersham Pharmacia Biotech) by the Megaprime DNA labeling system (Amersham Pharmacia Biotech). Using this DNA as a probe (probe 1),~1.6 × 106 plaques of a human liver cDNA library in [lambda]gt10 (Clontech) was screened by hybridization in Rapid hybridization buffer (Amersham Pharmacia Biotech) at 65°C. The filters were washed in 2 × SSPE/0.1% SDS at 65°C (Sambrook et al., 1989).

Transient expression of human GnT-IV in COS7 cells

The putative human GnT-IV cDNA was amplified by PCR using the sense primer 5[prime]-TTCTCGAGATGAGGCTCCGCAATGGAACTG-3[prime] (1-7F) and the antisense primer 5[prime]-AGAAATGTGGGCTTCAGGGCTGGC-3[prime] (1-7R) as shown in Figure 1. The amplified fragment was ligated into pCR-Script Amp SK(+) and its identity was confirmed by sequencing. A XhoI-SacI 1.7 kb insert containing GnT-IV cDNA was subcloned into an expression vector pSVL (Amersham Pharmacia Biotech) and designated pHGT4-1. Approximately 5 × 106 COS7 cells (RIKEN Cell Bank, Tsukuba Science City, Japan) in 0.8 ml of PBS(-) (Nissui Pharmaceutical, Tokyo, Japan) were mixed with 10 µg of plasmid DNA and transfected by electroporation at 1600 V and 25 µF on a Gene Pulser apparatus (Bio-Rad). The cells were then cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days at 37°C with 5% CO2. After harvesting the cells by scraping and centrifugation, the cell pellet was resuspended in 100 µl of 50 mM Tris-HCl (pH 7.5), 2 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication. The lysate was centrifuged by 2000 × g for 5 min at 4°C, and the resulting supernatant was assayed for GnT-IV activity.

Enzymatic assays for GnT-IV

The GnT-IV activity was measured by the method described by Oguri et al. (Oguri et al., 1997). The reaction velocitywas determined using GlcNAc[beta]1-2Man[alpha]1-6(GlcNAc[beta]1-2Man[alpha]1-3)Man[beta]1-4GlcNAc[beta]1-4GlcNAc[beta]1-PA, or Gn2(2',2)core-PA, as an acceptor. Protein quantity of the lysate was measured by Protein assay kit I (Bio-Rad) using bovine [gamma]-globulin as a standard protein.

Northern analysis

Northern blots containing ~2 µg of poly(A)+ mRNA from multiple human tissues (Human MTN, Human MTN II, Human MTN III, and Human Cancer Cell Line MTN) were obtained from Clontech. Human GnT-IV probe 2 (Figure 1) was labeled using the PCR Radioactive Labeling System (Life Technologies, Inc.) with [[alpha]-32P]dCTP (Amersham Pharmacia Biotech), the sense primer 5[prime]-GGCTATCACACCGATAGCTGGAG-3[prime] (1-8F), the antisense primer 5[prime]-TCCACCATTCCTTCTGCAACACC-3[prime] (1-9R), and pHGT4-1 as a template. The PCR reaction was performed for 30 cycles (30 s at 94°C, 75 s at 60°C, and 2 min at 72°C) after the initial denaturation for 10 min at 95°C on a Zymoreactor II (ATTO, Tokyo, Japan). Human GnT-IV probe 3 (Figure 1) was also radioactively labeled using this system except the sense primer 5[prime]-ACGTCTTCGAATAGCTGAAC-3[prime] (1-11F) and the antisense primer 5[prime]-TACTTGGCACTTGAAGAGAT-3[prime] (1-10R). A probe for human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was prepared using the same kit with the sense primer 5[prime]-CCAAAATCAAGTGGGGCGATG-3[prime] (G-1F) and the antisense primer 5[prime]-CAGGAGGCATTGCTGATGATCTTG-3[prime] (G-1R) (Arcari et al., 1984). The PCR reaction was carried out with an initial denaturation for 3 min at 95°C followed by 30 cycles (30 s at 94°C, 1 min at 55°C, and 1 min at 72°C). The blots were hybridized with each probe in Rapid hybridization buffer (Amersham Pharmacia Biotech) at 65°C, and were washed twice in 2 × SSPE/0.1% SDS and 0.1 × SSPE/0.1% SDS, respectively (Sambrook et al., 1989).

Isolation of a human GnT-IV genomic clone and chromosomal mapping

A partial genomic fragment encoding human GnT-IV was amplified by PCR using the sense primer 5[prime]-GTGGATCCAAGTCTCAATCCCAT-3[prime] (1-9F), the antisense primer 5[prime]-GTACAAGTCATATTAACAGGCTGTC-3[prime] (1-8R), and human genomic DNA (Clontech) as a template. The PCR was carried out for 35 cycles (15 s at 94°C, 15 s at 55°C, and 20 s at 72°C) after initial denaturation for 5 min at 95°C on a Gene Amp PCR System 9600. The amplified fragment was cloned into pCR-Script Amp SK(+) and sequenced. Using the same primer set, PCR screening of a human genomic P1 plasmid library was performed by Genome Systems. Purified DNA from one of the isolated P1 clones, clone F235, was labeled with digoxigenin-dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextransulfate and 2 × SSC. Specific hybridization signals were detected by incubating the hybridized slides in FITC-labeled anti-digoxigenin antibody followed by counterstaining with 4,6-diamino-2-phenylindole.

Acknowledgments

This work was performed as part of the Research and Development Projects of Industrial Science and Technology Frontier Program supported by New Energy and Industrial Technology Development Organization (NEDO). Mari T. Minowa is a Research Fellow of NEDO. We thank Drs. T. Taguchi and N. Taniguchi (Osaka University Medical School) for the GnT-VI assay and helpful suggestions, and Dr. M. Fukuda (Burnham Institute) for his suggestions regarding chromosomal mapping.

Abbreviations

GnT-IV, UDP-N-acetylglucosamine:[alpha]1,3-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase; 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-acetyl-glucosaminyltransferase; GnT-III, UDP-N-acetylglucosamine:[beta]1,4-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase; GnT-V, UDP-N-acetylglucosamine:[alpha]1,6-d-mannoside [beta]1,6-N-acetyl-glucosaminyltransferase; GnT-VI, UDP-N-acetylglucosamine:[alpha]1,6-d-mannoside [beta]1,4-N-acetylglucosaminyltransferase; [beta]1,4-GalT, UDP-galactose:N-acetylglucosamine [beta]1,4-galactosyl-transferase; hEPO, human erythropoietin; IL-6, interleukin 6; RT-PCR, polymerase chain reaction following reverse transcription of RNA; PA, pyridylaminated; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; FITC, fluorescein isothiocyanate.

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1To whom correspondence should be addressed
2Present address: Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan


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