In both plant and animal cells, a highly conserved multistep biosynthetic pathway covalently links carbohydrate to asparagine residues of newly synthesized proteins. This N-linked protein glycosylation is initiated in the endoplasmatic reticulum (ER) by the transfer of Glc3Man9GlcNAc2 glycans to asparagine residues in the sequence Asn-X-Ser/Thr of the nascent polypeptide. Glycosidases and glycosyltransferases in the ER and in the Golgi compartment subsequently convert typical Glc3Man9GlcNAc2 first to oligo mannose glycans (Man5GlcNAc2) and further to complex and hybrid glycans. The first step that initiates the formation of hybrid or complex N-glycans in both plants and animals is catalyzed by the Golgi enzyme [beta]1,2N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) (for a review, see Kornfeld and Kornfeld, 1985). Enzymes that act beyond this point (e.g., [alpha]-mannosidase II, GlcNAc-TII) require prior GlcNAc-TI action. Shortly beyond GlcNAc-TI activity, the pathways for complex N-glycan biosynthesis in plants and animals begin to diverge. Whereas oligo mannose glycans have the same structure in plant and in animal cells, complex glycans of plants are generally smaller and contain [beta]1-2 xylose and/or [alpha]1-3 fucose residues attached to the Man3-5GlcNAc2 core which are with a few exceptions (van Kuik et al., 1985; Kubelka et al., 1993; Mulder et al., 1995) not present on animal glycoproteins.
Although the biosynthesis of N-glycans in plants and animals is basically the same (for review, see Fitchette-Lainè, 1998) several GlcNAc-TI genes from different animal species have been characterized (mammals: Kumar et al., 1990; Sarkar et al., 1991; Pownall et al., 1992; Fukada et al., 1994; Puthalakath et al., 1996; C.elegans: Wilson et al., 1994; Schachter et al., 1997; X.laevis: Mucha et al., unpublished observations), but so far no genes responsible for glycosyltransferases have been identified in plants. However, Arabidopsis thaliana mutants, lacking GlcNAc-TI activity were isolated using random mutagenesis (von Schaewen et al. 1993). The glycoproteins in such mutants have an abundance of Man5GlcNAc2 oligo mannose type N-glycans.
All characterized GlcNAc-TI genes code for a type II transmembrane protein with a globular C-terminal part containing the catalytic domain. This domain is well conserved between different species and its activity can be complemented even between species with a great evolutionary distance. For example, GlcNAc-TI activity from GlcNAc-TI deficient Arabidopsis thaliana plants can be complemented by transient expression of human GlcNAc-TI (Gomez and Chrispeels, 1994).
We have cloned the cDNA putatively encoding the enzyme [beta]1,2N-acetylglucosaminyltransferase I from tobacco. In the present paper we show the nucleotide sequence of the cDNA, enzymatic activity of the putative catalytic protein domain and the expression of GlcNAc-TI mRNA in tobacco leaves.
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Figure 1. (a) Sequence of the tobacco cDNA coding for GlcNAc-TI including 5[prime]- and 3[prime]-nontranslated regions. The deduced amino acid sequence starts at nucleotide position 77 and ends at position 1414, the termination codon is marked (*). The putative transmembrane region and the potential N-glycosylation site are underlined. (b) Amino acid sequence alignment of the GlcNAc-TI C-terminus from tobacco, human, and extracted sequence from an A.thaliana BAC clone (EMBL-Nr: B24856). Numbering refers to the amino acid position of the respective clones and represent part of the catalytic domain of the enzymes. N.t., N.tabacum; H.s., H. sapiens; A.th., A. thaliana. Consensus sequence is shaded. Identification of the cDNA coding for GlcNAc-TI from Nicotiana tabacum
Employing degenerated primers (pnew1 and pnew3) representing highly conserved regions of known GlcNAc-TI genes from animals we were able to amplify a 93 bp fragment from tobacco leaf cDNA. Sequence information of this fragment revealed homology to parts of the catalytic region of known GlcNAc-TI genes (Kumar et al., 1990; Sarkar et al., 1991; Pownall et al., 1992; Fukada et al., 1994; Puthalakath et al., 1996). This PCR fragment was used as a homologous probe to screen for a corresponding cDNA clone coding for GlcNAc-TI in a N.tabacum [lambda] phage cDNA library. Upon screening of about 0.5 × 106 plaques one positive clone was found (5/2). The insert from the phage was obtained by in vivo excision, subcloned, and nucleic acid sequence was determined. The sequence of the clone revealed an open reading frame of 1338 nucleotides and allowed the prediction of a 446 amino acid protein with a molecular weight of 52.1 kDa (Figure Characterization of the deduced amino acid sequence and sequence homology to GlcNAc-TI proteins of mammals
Hydrophobicity analysis (Kyte and Doolittle, 1982) of the deduced amino acid sequence of GlcNAc-TI cDNA revealed a hydrophobic domain near the amino-terminus (residues 12-26, underlined in Figure
Amino acid sequence alignment of GlcNAc-TI from tobacco and mammals revealed no homology considering the putative cytoplasmic, transmembrane and stem region (CTS) (Sarkar et al., 1998). However, at the putative catalytic domain from GlcNAc-TI (Sarkar et al., 1998) several single amino acids and peptide motifs were found to be conserved in human and tobacco GlcNAc-TI. For example, from amino acid position 184 to 349 65 amino acid residues (40%) were found to be conserved between the two species (Figure
GenBank and Swissprot database searches did not reveal significant sequence similarities to known DNA or protein sequences except with those of previously identified GlcNAc-TI sequences. However, searching an Arabidopsis thaliana database (http://genome-www2.stanford.edu/cgi-bin/AtDB/nph-blast2atdb) a small portion of the tobacco cDNA sequence (3[prime]-end) matched parts of the sequence from a genomic Arabidopsis thaliana BAC clone (EMBL-Nr: B24856). A.thaliana sequence was extracted, and subsequently three putative introns were excluded from the sequence. After reverse complementation and translation of the sequence, we were able to reveal an amino acid sequence homology of 72% between the A.thaliana and the tobacco clone within 121 amino acid residues (Figure
Figure 2. Demonstration of GlcNAc-T1 activity. The pyridylaminated substrates. Man5GlcNAc2 or Man3GlcNAc2 were incubated with lysate of baculovirus infected cells in the presence of UDP-GlcNAc for 4.5 h. Subsequently, substrate (S) and GlcNAc-TI product (P) were separated by reversed-phase HPLC. Analysis of cells infected with recombinant baculovirus expressing truncated GlcNAc-TI cDNA using Man5GlcNAc2-PA (a) and Man3GlcNAc2-PA (b); (c) and (d), analysis of mock infected cells using Man5GlcNAc2-PA and Man3GlcNAc2-PA, respectively. S, Substrate; P, product.
The deduced amino acid sequence further contains one potential N-glycosylation site at position 203 having the consensus sequence N-F-S. However, GlcNAc-TI polypeptides from animals are usually not N-glycosylated (Kumar et al., 1990; Sarkar et al., 1991). Whether tobacco GlcNAc-TI is actually N-glycosylated in vivo remains to be determined. Expression of the putative catalytic domain of GlcNAc-TI cDNA in insect cells and determination of enzyme activity
A recombinant baculovirus was constructed to express the putative catalytic domain of the tobacco GlcNAc-TI in Spodoptera frugiperda (Sf9)-cells. Since the catalytic domain of GlcNAc-TI is sufficient for enzyme activity (Sarkar et al., 1998; Mucha et al., unpublished observations), a virus transfer vector was designed for the expression of a truncated GlcNAc-TI -GST fusion protein, lacking the putative cytoplasmic, transmembrane, and stem region. In order to examine whether this truncated GlcNAc-TI form is enzymatically active, the lysate and culture media from cells infected with the recombinant baculovirus were subjected to an in vitro enzyme activity assay using pyridylaminated Man3GlcNAc2 or Man5GlcNAc2 as acceptor substrate. The activity of recombinant GlcNAc-TI was higher when physiological Man5GlcNAc2 was used (Figure Copy number determination of the putative GlcNAc-TI gene and its expression in leaves
In order to determine the genomic copy number for the GlcNAc-TI gene Southern blot analysis was carried out (Figure
Figure 3. Southern blot analysis. Genomic DNA (12 µg) from N.tabacum (1) and N.benthamiana (2) was digested with HindIII, separated on an agarose gel, blotted, and hybridized with two nonoverlapping fragments of GlcNAc-TI cDNA. The blot was hybridized with probe 1 (a), stripped, and subsequently hybridized with probe 2 (b). The black bar represents the full-length GlcNAc-TI cDNA clone; H, HindIII restriction site; the DNA length standard is given in base pairs. The weak signal at position 4000 in N.tabacum (b) is assumed to be a stripping artefact originating from the first hybridization.
In order to verify the expression of GlcNAc-TI mRNA in tobacco leaves RT-PCR was carried out. Total RNA from leaves were isolated and cDNA was obtained using a specific GlcNAcTI primer located near the 3[prime]-end of the gene. Subsequently a PCR amplification of the cDNA using GlcNAcTI specific primers was carried out (Figure
Figure 4. Detection of GlcNAc-TI mRNA expression by reverse transcription (RT)-PCR. Total RNA isolated from tobacco leaves served as template for the RT using a GlcNAc-TI specific primer. For the subsequent PCR reaction GlcNAc-TI specific primers were used. The expected size of the amplified PCR product is 470 bp. M, Standard in base pairs; (1), negative control (no mRNA but RT and PCR were carried out); (2), no RT (only PCR reaction from the RNA template was carried out); (3), RT-PCR with RNA template.
We have isolated a cDNA clone from a N.tabacum cDNA library representing the complete coding sequence for GlcNAc-TI. It contains an open reading frame of 1338 nucleotides that encodes a polypeptide of 446 amino acids, with a calculated molecular mass of the polypeptide of 52.1 kDa. Although 5[prime]-noncoding regions from animal GlcNAc-TI cDNAs range from 158 (rat) to 587 nucleotides (frog), we were not able to identify more than 76 nucleotides in the corresponding tobacco cDNA. However, the 5[prime]-noncoding sequence probably extends further upstream.
In summary 0.5 × 106 phage colonies were screened but only one positive clone containing the tobacco GlcNAc-TI sequence was detected. For some animals several 'isoforms" of GlcNAc-TI have been identified on the cDNA but so far not on the protein level. For example in C.elegans (Schachter et al., 1997) and in X. laevis (Mucha et al., unpublished observations) three and two cDNA 'isoforms," respectively, which differ in several amino acid residues in their deduced amino acid sequence were found. Our data do not provide evidence that more than one isoform of GlcNAc-TI exists in tobacco.
Analysis of the deduced amino acid sequence of the tobacco GlcNAc-TI protein predicts a functional domain architecture and type II transmembrane topology typical of the glycosyltransferases that have been cloned to date. The native form of tobacco GlcNAc-TI consists obviously of a short (11 amino acids) N-terminal cytoplasmic segment, a 15-amino acid hydrophobic stretch, which should act as a membrane anchor and a large C-terminal luminal region where the stem region and the catalytic domain are located. The amino acid sequence further contains one potential N-glycosylation site. Although GlcNAc-TI polypeptides from animals are usually not N-glycosylated (Kumar et al., 1990; Sarkar et al., 1991) there is some evidence that GlcNAc-TII isolated from rat is N-glycosylated at one site (DAgostaro et al., 1995) and for rat GlcNAc-TIII it has been shown that N-glycosylation is a prerequisite for enzyme activity and Golgi retention (Nagai et al., 1997). Whether tobacco GlcNAc-TI is actually N-glycosylated and whether this has an effect for the enzyme was not investigated.
A detailed sequence homology analysis of the tobacco and the mammalian GlcNAc-TI polypeptide revealed 40% sequence homology within the catalytic domain. Although little information about the importance of these conserved residues is available, it has been demonstrated that single point mutations in this well conserved area can destroy enzyme activity (Puthalakath et al., 1996).
An extensive search of the Arabidopsis thaliana genomic DNA sequence database revealed regions of significant sequence homology to one end of a BAC clone. Our data strongly indicate that the genomic sequence of A.thaliana represents a portion of the GlcNAc-TI gene. Although GlcNAc-TI genes from mammals do not have introns there is evidence that GlcNAc-TI genes from C.elegans have an exon/intron structure (Schachter et al., 1997). Putative introns have been found within the sequence which may represent parts of the A.thaliana GlcNAc-TI gene. It remains to be investigated whether the tobacco GlcNAc-TI gene contains introns since the genomic structure of the tobacco gene has not been investigated.
The conversion of both substrates Man5GlcNAc2-PA and Man3GlcNAc2-PA by the recombinant tobacco GlcNAc TI demonstrates that the gene for the active enzyme has been cloned. The identity of the product has been confirmed both by HPLC and MALDI-TOF. In general it has been shown that glycosyltransferases have a precise acceptor and donor substrate specificity. GlcNAc-TI from mammals uses the physiological acceptor substrate Man5GlcNAc2-R and the artificial substrate Man3GlcNAc2-R with a similar transfer rate. However, in insects GlcNAc-TI displays a much higher preference for the physiological substrate (Altmann et al., 1993). Similarly, the tobacco GlcNAc-TI exhibited higher activity when using the Man5GlcNAc2 substrate. However, kinetic parameters have not been determined so far.
In N.tabacum as well as in N.benthamiana two hybridization signals were detected in Southern blot analyses using two independent probes. Since N.tabacum is amphidiploid with 2n=4x=48 (Kenton et al., 1993; Moscone et al., 1996), it is very likely that the two hybridization signals are derived rather from the two genomes than from two gene copies. However, the amphidiploid origin of N.benthamiana has not been proven, and hence it remains unclear whether the signals that were obtained represent two copies of the gene or one copy from the two genomes. While mammalian GlcNAc-TI is present as a single copy gene (e.g., Schachter et al., 1997), in C.elegans three genes homologous to GlcNAc-TI that putatively code for three isoforms were detected (Schachter et al., 1997). Our results suggest that in tobacco, as in mammals, GlcNAc-TI is represented by a single copy gene. Plant material
N.tabacum and N.benthamiana plants were cultivated in a controlled growth chamber with 22°C day and night temperature, a 16 h photoperiod, and 50% humidity. Cloning of a homologous GlcNAc-TI cDNA fragment from N.tabacum
Total RNA was isolated from newly emerging N.tabacum leaves using the TRIzol RNA extraction kit (Life Technologies). First-strand cDNA was synthesized from 1.8 µg of total RNA primed by an oligo(dT)15 primer using the Reverse Transcription System (Promega). Regions of conserved amino acid sequence within the catalytical domain from known GlcNAc-TI were used to design degenerated primers pnew1 (position 791-816 from tobacco GlcNAc-TI), 5[prime]-G(C,T)GTITCIGCITGGAA (C,T)GA(C,T)AA- (C,T)GG-3[prime] (I = Inosine), and primer pnew3 (position 860-883), 5[prime]-CCAICCIAGICCIG (C,G)(A,G)AA(A,G)AA(A,G)TC-3[prime]. PCR amplification was carried out with an initial denaturation step at 94°C for 4 min, followed by 40 amplification cycles: 94°C for 1 min, 52°C for 2 min, and 72°C for 2 min. After completion of 40 cycles, the reaction mixture was maintained at 72°C for 8 min. The resulting PCR product was extracted from an agarose gel using QIAEX II (QIAGEN) and blunt-end cloned into SmaI linearized pUC19 (Pharmacia Biotech) plasmid. cDNA library screening
Approximately 500,000 pfu of a cDNA library from Nicotiana tabacum leaf in [lambda]ZAPII (Stratagene) were blotted on nylon membranes (Boehringer Mannheim) and screened using the PCR product obtained with pnew1/pnew3 primers as a probe. ThePCR fragment was labeled with [alpha]-32P-dCTP using an Oligolabelling kit (Pharmacia Biotech). Filters were hybridized according to Church and Gilbert, (1984) at 65°C overnight. Washing was performed in 2× SSC, 0.1 % SDS at 60° C and in 0.5× SSC, 0.1 % SDS at 55°C. Plaque-purified positive clones were converted into phagemids (Bluescript SKII) by in vivo excision from [lambda]ZAPII following the manufacturer's protocol (Stratagene). 5[prime] RACE amplification of cDNA
The 5[prime]-terminus of the N.tabacum GlcNAc-TI cDNA was amplified using the 5[prime] RACE System kit (Life Technologies) according to the supplier's recommendations. Gene-specific primer tab2r (pos. 836-860) was used in first-strand synthesis and the PCR amplification of the cDNA was performed using tab9r (pos. 410-430) and tab3r (pos. 315-332). RT-PCR
Total RNA isolation from tobacco leaves and reverse transcriptase reaction were performed as described above. PCR amplification was carried out using primer tab4f (pos. 391-412) and primer tab2r with an initial denaturation step at 94°C for 3 min, followed by 28 amplification cycles: 94°C for 45 sec, 50°C for 1 min, and 72°C for 1 min 30 sec. After completion of 28 cycles, the reaction mixture was maintained at 72°C for 8 min. Southern hybridizations
Genomic DNA was isolated from N.tabacum and N.benthamiana leaves using the DNeasy Plant kit (QIAGEN), digested with Hind III and separated on 0.8% agarose gel. Transfer of the DNA fragments to nylon membranes (Boehringer Mannheim) were performed under neutral conditions (20× SSC). Hybridization and washing was performed as described above. Two nonoverlapping PCR fragments corresponding to GlcNAc-TI cDNA were used as probes: probe one was obtained with primer tab1f (pos. 823-845) and Saltab5r (pos.1401-1420); probe two was obtained using primers Saltab11f (pos. 77-96) and tab9r. DNA sequencing
QIAGEN plasmid kit (QIAGEN) or a Flexiprep kit (Pharmacia Biotech) purified DNA was used for DNA sequencing reactions. Cycle sequencing according to the recommendations of the manufacturer (Applied Biosystems) was carried out. DNA and protein sequence analysis was done using DNASTAR PC-software. Expression in insect cells
The putative catalytic domain of GlcNAc-TI cDNA was amplified with primers Bamtab4f (pos. 392-410) and Ecotab5r (pos. 1401-1420) using pBluescript-SK containing the whole GlcNAc-TI coding region (clone 5/2) as a template. The oligonucleotides contain a BamHI, respectively, an EcoRI restriction site and the PCR product hence ligated into BamHI-EcoRI sites of the baculovirus transfer vector pAcSecG2T (PharMingen). The truncated gene was cloned downstream from and in frame with GST in order to produce a recombinant fusion protein. The correctness of the cloning product was confirmed by sequencing. Sf-9 cells were grown in serum-free IPL-41 medium (Sigma) at 27°C; 1 µg of recombinant baculovirus transfer vector was cotransfected with 200 ng linear Baculo-Gold DNA (PharMingen) into 1 × 106 Sf-9 cells/60 mm tissue-culture dish using Lipofectin (Life Technologies). Cells were incubated for 5 days at 27°C. The supernatant was used to infect 2 × 106 Sf-9 cells/60 mm tissue-culture dish in IPL-41 medium supplemented with 5% fetal calf serum. Cells were incubated for 4 days at 27°C. GlcNAc-TI assay and characterization of product
GlcNAc-TI activity was determined by reversed phase HPLC using either pyridylaminated Man5GlcNAc2 or Man3GlcNAc2 as the acceptor substrate and culture supernatant or homogenized cells as the enzyme source (Altmann et al., 1993). Mock infected cells were used as controls. MALDI-TOF mass spectrometry and exoglycosidase digestions were done as previously described (Wilson and Altmann, in press). Core [alpha]1,6-fucosyltransferase from porcine brain was partially purified according to Uozumi et al., (1996). Fucosyltransfer was measured as described by Staudacher et al., (1991). Digestion with jack bean N-acetyl-[beta]-glucosaminidase (Sigma) was performed as described previously (Kubelka et al., 1993).
We thank Eva Turetschek and Barbara Svoboda for excellent technical support. These studies were supported by a grant from the EC Project BIO4CT 960304.
GlcNAc-T, N-acetylglucosaminyltransferase; GST, glutathione S-transferase; -PA, -pyridylamine; pfu, plaque forming units; RACE, rapid amplification of cDNA ends; SDS, sodium dodecyl sulfate; SSC, sodium chloride-sodium citrate; UDP-GlcNAc, UDP-N-acetylglucosamine.
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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