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).
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
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.
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
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
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
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).
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
Table II.
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
The myeloid cell line HL-60 shows high expression of GnT-IV in contrast to myeloblastic lymphoma cell line K-562 (Figure
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
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. 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 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 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 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.
Discussion
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
Materials and methods
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.
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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