Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:{alpha}3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I

Jan Mucha2, Barbara Svoboda2, Ulrike Fröhwein2, Richard Strasser2, Manfred Mischinger2, Herwig Schwihla2, Friedrich Altmann3, Werner Hane4, Harry Schachter5, Josef Glössl2 and Lukas Mach1,2

2Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria; 3Institut für Chemie, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Vienna, Austria; 4Ernst-Boehringer-Institut für Arzneimittelforschung, Bender & Co GesmbH, Dr. Boehringer-Gasse 5–11, A-1121 Vienna, Austria; and 5Department of Biochemistry, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada

Received on April 24, 2001; revised on June 11, 2001; accepted on June 14, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
UDP-GlcNAc:{alpha}3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoenzyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.

Key words: GlcNAc-transferase I/glycosyltransferase/N-glycan/biosynthesis/amphibian


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A highly conserved multistep biosynthetic pathway leads to the covalent modification of newly synthesized proteins with N-glycosidically linked oligosaccharides. The process of N-linked protein glycosylation is initiated in the endoplasmic reticulum (ER) by the transfer of a Glc3Man9GlcNAc2 precursor to asparagine residues in the sequence Asn-X-Ser/Thr of the nascent polypeptide. This glycan is the subject of extensive trimming by glycosidases in the ER and in the Golgi apparatus, resulting in the formation of oligomannosidic N-linked oligosaccharides. The latter structures undergo further processing in the Golgi apparatus and are ultimately converted into N-glycans of the complex type (for review see Kornfeld and Kornfeld, 1985Go).

The biosynthesis of complex-type N-glycans involves a large array of Golgi-resident glycosyltransferases. The first key enzyme in the conversion of oligomannosidic into complex-type structures is UDP-GlcNAc:{alpha}3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101). Mice lacking this enzyme display severe developmental defects and die in utero, indicating a critical role of complex-type N-glycans in mammalian morphogenesis and development (Ioffe and Stanley, 1994Go; Metzler et al., 1994Go). Proper action of GnTI is a strict prerequisite for the in vivo activity of several other enzymes contributing to N-glycan processing, including UDP-GlcNAc:{alpha}6-D-mannoside ß-1,2-N-acetylglucosaminyltransferase II. It is noteworthy that in humans deficiency of the latter enzyme causes congenital disorder of glycosylation type IIa (previously called carbohydrate-deficient glycoprotein syndrome type II), an inherited disorder with defective brain development (Tan et al., 1996Go).

Most GnTI cDNA sequences available to date were obtained from human and rodent sources (Kumar et al., 1990Go; Sarkar et al., 1991Go; Pownall et al., 1992Go; Fukada et al., 1994Go; Puthalakath et al., 1996Go; Opat et al., 1998Go). Only one GnTI gene was found in all mammalian species studied so far. However, three GnTI genes are present in the genome of the nematode Caenorhabditis elegans, and the corresponding cDNAs were recently isolated and characterized (Chen et al., 1999Go). Concomitantly, we and others reported the molecular cloning of GnTI cDNAs from the plants Nicotiana tabacum (Strasser et al., 1999Go) and Arabidopsis thaliana (Bakker et al., 1999Go). All GnTI forms are type II transmembrane proteins and consist of a short N-terminal cytoplasmic tail, a single transmembrane anchor, a luminal stem region and a large C-terminal catalytic domain. The latter part of the enzyme is particularly well conserved through evolution, and its activity can be readily complemented even between species separated by a long evolutionary distance. For example, the phenotype of GnTI-deficient A. thaliana plants was reversed by ectopic expression of the human enzyme (Gomez and Chrispeels, 1994Go). Vice versa, the glycosylation defect of Chinese hamster ovary (CHO) Lec1 cells that lack endogenous GnTI activity was abrogated by transfection with an A. thaliana GnTI cDNA (Bakker et al., 1999Go).

Oocytes of the clawed frog Xenopus laevis are frequently used for the production of recombinant proteins. The system is particularly well suited for functional screening of large cDNA pools and rapid analysis of site-directed mutations. However, information on the N-glycosylation potential of these cells is scarce beyond limited evidence that X. laevis oocytes synthesize both oligomannosidic and complex-type N-glycans (Roitsch and Lehle, 1989Go; Cantor and Kornfeld, 1992Go). We now report that the genome of X. laevis encodes two closely related GnTI isoenzymes that are both catalytically active and appear to occur in all frog tissues.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation and analysis of X. laevis GnTI cDNAs
Polymerase chain reaction (PCR) analysis of genomic X. laevis DNA with degenerate oligonucleotide primers based on highly conserved regions of known animal GnTI cDNAs yielded a 393-bp product with striking similarity to previously elucidated GnTI sequences. This fragment was used to isolate five GnTI cDNA clones from 5 x 106 plaques of a X. laevis ovary {lambda} phage cDNA library. DNA sequencing indicated that these clones were derived from two closely related but clearly distinct genes. Three clones contained an open reading frame for a protein of 448 amino acids with a calculated molecular mass of 51.1 kDa (isoenzyme A). The other two cDNAs were found to encode a polypeptide with 444 residues and a predicted size of 50.6 kDa (isoenzyme B). The C-terminal regions of both proteins display a high extent of similarity to mammalian, plant, and C. elegans GnTI sequences. In both cDNA sets, the sequence context of the first ATG at position 572–574 (isoenzyme A) and 587–589 (isoenzyme B) is favorable for initiation of translation. Hence, we consider these triplets to represent the respective start codons. While the 5'-untranslated regions are closely related, the respective 3'-noncoding cDNA sequences lack any significant similarities. The 3'-untranslated region of the cDNA encoding isoenzyme B comprises 970 base pairs prior to a poly(A) stretch. A polyadenylation signal identical to the animal consensus sequence AATAAA was identified at positions 2479 to 2484. Despite their considerable length (2.2–2.5 kb), none of the cDNAs representing isoenzyme A contained a poly(A) tail or a potential polyadenylation signal, indicating that the original size of its transcript exceeds 2500 bases. The coding regions of the genes for isoenzymes A and B are not interrupted by introns, as demonstrated by PCR analysis of genomic X. laevis DNA (data not shown).

Hydropathy plot analysis of the deduced amino acid sequences indicated for both isoenzymes a putative transmembrane domain at residues 5–26. This region is capped at the amino terminus by a short hydrophilic segment comprised of four amino acids, probably protruding into the cytoplasm. The luminal part of each protein consists of a stem region (81 and 77 residues for isoenzymes A and B, respectively) and the carboxy-terminal catalytic domain (341 amino acids). Thus, X. laevis GnTI exhibits a type II transmembrane protein topology as typical for glycosyltransferases. Amino acid sequence alignment with its mammalian, plant, and C. elegans orthologues revealed no pronounced similarity within the putative cytoplasmic, transmembrane, and stem (CTS) regions. The stem regions of isoenzymes A and B vary not only in length but also in 13 individual residues. Furthermore, the amino acid sequences of their catalytic domains differ at 18 positions. However, all residues strictly conserved across other species and hence possibly critical for proper function of the enzyme are preserved in X. laevis GnTI. GenBank and SwissProt database searches did not reveal significant homologies to known DNA or protein sequences other than to previously cloned GnTI forms. The catalytic domain of X. laevis GnTI displays 67%, 37%, and 30% sequence identity to its counterparts from humans, C. elegans, and N. tabacum, respectively (Figure 1).



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Fig. 1. Amino acid sequence alignments of X. laevis GnTI isoenzymes A and B with their human, nematode and plant counterparts. Residues strictly conserved within all species are given on gray background. Dashes indicate gaps. The asterisk (*) marks the position of Thr223 in isoenzyme B. The first residues of the catalytic domain are highlighted. The catalytic domain starts at Val108 (X.l.A), Val104 (X.l.B), Val105 (H.s.), Val107 (N.t.), and Val71 (C.e.), respectively (Sarkar et al., 1998Go; Chen et al., 1999Go). X.l.A, X. laevis GnTI isoenzyme A; X.l.B, X. laevis GnTI isoenzyme B; H.s., Homo sapiens GnTI, N.t., N. tabacum GnTI; C.e., C. elegans GnTI (isoenzyme gly-12).

 
Heterologous expression of X. laevis GnTI cDNAs in insect cells
Recombinant baculoviruses were produced to permit the expression of X. laevis GnTI cDNAs in Spodoptera frugiperda Sf21 cells. Truncated versions lacking the CTS regions were fused to the carboxy terminus of Schistosoma japonicum glutathione S-transferase (GST) and inserted into the viral genome by homologous recombination. These constructs were introduced into Sf21 cells for expression under control of the polyhedrin promoter. Because the GnTI-GST fusion protein contains a signal peptide for targeting to the secretory pathway, supernatants as well as lysates of cells infected with the recombinant baculoviruses were analyzed for GnTI activity in vitro. Pyridylaminated Man3GlcNAc2 was chosen as acceptor substrate because Sf21 GnTI exhibits, in contrast to its mammalian counterparts, only negligible activity toward this compound (Altmann et al., 1993Go). With this substrate, the GnTI activity of homogenates from Sf21 cells infected with the parental baculovirus (mock-infected cells) was barely detectable (< 0.2 nmol of product formed per h per mg of total cellular protein), and the corresponding conditioned culture medium was devoid of any measurable enzymatic activity. In contrast, lysates of Sf21 cells expressing X. laevis GnTI isoenzyme A were highly active (34 nmol of product formed per h per mg of total cellular protein). However, the main fraction of the recombinant enzyme was found in the culture supernatant (98 nmol of product formed per h per mg of total cellular protein). These results demonstrate that isoenzyme A displays significant catalytic activity and can be efficiently produced at high levels in insect cells as a secreted, soluble protein.

The GST-GnTI protein content of the samples was estimated by immunoblotting with antibodies to GST and purified recombinant GST as a standard. The amount of total GST-GnTI produced was found to be about 1 mg per L of culture, which compares favorably with previously reported data on the expression of a truncated form of rabbit GnTI in insect cells (Sarkar, 1994Go). The calculated specific activity of cell-associated isoenzyme A at the nonsaturating conditions used in our GnTI activity assays was 0.6 µmol of product formed per min per mg of enzyme protein, somewhat lower than that observed for insect cell–derived rabbit GnTI under similar conditions (2 µmol of product formed per min per mg of enzyme protein; Sarkar, 1994Go). The Km(app) value for pyridylaminated Man3GlcNAc2 was estimated to be 1.6 mM at an initial concentration of UDP-GlcNAc of 2 mM, which is within the same range as determined for the recombinant rabbit enzyme (1.0 mM; Sarkar, 1994Go).

On expression of the catalytic domain of X. laevis GnTI isoenzyme B in Sf21 cells, neither the conditioned culture medium nor the lysate of the infected cells exhibited appreciable enzymatic activity with pyridylaminated Man3GlcNAc2 as substrate (< 0.2 nmol of product formed per h per mg of total cellular protein). However, the recombinant fusion protein was synthesized at a level comparable with isoenzyme A, as judged by immunoblotting with anti-GST antibodies. Furthermore, the polypeptide was of the expected size (65 kDa), thus effectively ruling out that partial proteolysis had occurred (Figure 2A). Expression of the entire coding region of isoenzyme B cDNA in Sf21 cells also failed to yield any significant intracellular GnTI activity with pyridylaminated Man3GlcNAc2 (data not shown).



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Fig. 2. Heterologous expression of X. laevis GnTI cDNAs in insect cells. (A) Lysates of Sf21 cells expressing the catalytic domains of isoenzymes A (lane 3) and B (lane 2) as GST fusion proteins were analyzed by immunoblotting with antibodies to GST as outlined in Materials and methods. Purified GST (25 ng) was used as a control (lane 1). The migration positions of bovine serum albumin (66 kDa), chicken ovalbumin (45 kDa), and bovine carbonic anhydrase (29 kDa) are indicated. The observed size of the recombinant polypeptides (65 kDa) was in close agreement with their theoretical molecular masses. No reaction was observed with lysates of uninfected Sf21 cells. (B and C) Lysates (C) and supernatants (M) of Sf21 cells expressing the catalytic domains of wild-type and mutant isoenzymes A (C) and B (B) fused to a heterologous leader sequence were analyzed for GnTI activity (expressed in nmol of product formed per h per mg of total protein), using Man3-octyl as the acceptor substrate. The GnTI protein content of the samples was assessed by immunoblotting with antibodies to the enterokinase cleavage site present in the recombinant proteins and is given as the percentage of the total amount of GnTI protein detected in the individual cultures. Sample amounts were in each case equivalent to 103 cells. The migration positions of prestained protein standards are indicated. The observed size of the recombinant polypeptides (38 kDa) is in close agreement with their theoretical molecular masses. No specific signals were obtained with lysates and conditioned media of uninfected Sf21 cells.

 
As an alternative approach to achieve production of a secreted, soluble variant of isoenzyme B, the catalytic domain of the enzyme was fused to a leader sequence containing a cleavable signal peptide, a hexahistidine tag, and an enterokinase cleavage site. This construct was inserted into the baculovirus genome, and the recombinant viruses thus generated were used to infect Sf21 cells. For comparison, isoenzyme A was expressed in the same manner. A radiometric GnTI assay using Man3-octyl as acceptor substrate was used in these experiments (Altmann et al., 1993Go). When lysates of uninfected Sf21 cells were assayed with this substrate, considerable endogenous GnTI activity was detected (1.8 nmol of product formed per h per mg of total cellular protein). The corresponding culture supernatant was devoid of any measurable enzymatic activity.

In contrast, culture medium conditioned by Sf21 cells expressing isoenzyme B contained significant amounts of GnTI activity (106 nmol of product formed per h per mg of total cellular protein). The enzymatic activity of the corresponding homogenate was only slightly elevated as compared to the control lysate (2.5 nmol of product produced per h per mg total cellular protein), indicating that > 95% of the functional enzyme was secreted into the culture supernatant. However, immunoblotting with antibodies to the enterokinase cleavage site revealed that the cell lysate contained the major fraction (91%) of the recombinant protein (Figure 2B). This suggests that isoenzyme B tends to be incorrectly folded, leading to the retention of inactive material within the cells. When Sf21 cells expressing isoenzyme A were analysed, a considerably larger part of the recombinant protein (37%) was secreted into the culture supernatant (Figure 2C). In agreement with this observation, the enzymatic activity of the conditioned culture medium was higher (199 nmol of product produced per h per mg of total cellular protein). These results support the notion that X. laevis GnTI isoenzyme B, though catalytically active, is inefficiently synthesized in vivo.

Substitution of Thr223 with Ala confers X. laevis GnTI isoenzyme B with properties reminiscent of isoenzyme A
The catalytic domains of X. laevis GnTI isoenzymes A and B differ at 18 positions. Isoenzyme B amino acids Thr223 and Ser226 were selected as targets for site-directed mutagenesis because the corresponding residues Ala227 and Pro230 of isoenzyme A are also invariably present in all available mammalian GnTI sequences. Two nucleotides were changed in isoenzyme B cDNA to create codons for Ala223 and Pro226, and the sequence encoding the respective mutant catalytic domain was expressed as a GST fusion construct in insect cells. Lysates of baculovirus-infected Sf21 cells displayed significant GnTI activity (44 nmol of product formed per h per mg total cellular protein). The Km(app) value for pyridylaminated Man3GlcNAc2 was estimated to be 3.0 mM at an initial concentration of UDP-GlcNAc of 2 mM, which is only slightly higher than observed for isoenzyme A under the same conditions. When Thr223->Ala and Ser226->Pro were separately introduced, Ala223 was identified as the critical residue, whereas Pro226 had no detectable impact (data not shown).

To confirm that Thr223 was indeed responsible for the observed accumulation of inactive isoenzyme B within the cells, this amino acid was introduced into isoenzyme A at the corresponding position (Ala227). The catalytic domain of this variant of isoenzyme A was then expressed as a hexahistidine-tagged fusion protein in Sf21 cells. In parallel, a mutant form of isoenzyme B containing Thr223->Ala was generated in the same manner. In contrast to its wild-type counterpart, isoenzyme A (Ala227->Thr) was not secreted into the culture medium (Figure 2C). Furthermore, the intracellular GnTI activity (1.7 nmol of product formed per h per mg of total cellular protein) was essentially the same as that of uninfected Sf21 cells, despite the presence of significant amounts of the recombinant protein. These results support the conclusion that replacement of Ala227 with Thr interferes with the synthesis of functional isoenzyme A. When Sf21 cells producing mutant isoenzyme B (Thr223->Ala) were analyzed, it was observed that a significantly larger fraction of the recombinant protein (30%) was secreted as compared with the corresponding wild-type form (9%; Figure 2B). Concomitantly, the GnTI activity of the culture supernatant was considerably increased (328 nmol of product formed per h per mg of total cellular protein). Intriguingly, the yield of functional isoenzyme B (Thr223->Ala) was similar to wild-type isoenzyme A produced under the same conditions, as based on comparison of both the GnTI activity and the recombinant protein content of the respective culture supernatants.

To directly compare the enzymatic capacity of wild-type and mutant isoenzyme B, the recombinant proteins were isolated from the culture supernatants of Sf21 cells infected with the respective baculoviruses by virtue of their hexahistidine tags. Although the yield of wild-type isoenzyme B was lower than that of its mutant variant, the specific GnTI activity of the two purified proteins was almost identical (251 mU/mg and 272 mU/mg of protein, respectively, when assayed with Man3-octyl as acceptor substrate). Furthermore, these properties compare well with the features of wild-type isoenzyme A (219 mU/mg of protein) purified and analyzed by the same procedure.

These results demonstrate that replacement of a single amino acid (Thr223->Ala) is sufficient to confer isoenzyme B with properties similar to isoenzyme A. Thr223 does not interfere with the catalytic mechanism of the enzyme but appears to impair proper assembly of the protein into an enzymatically competent state. However, reverse transcriptase (RT)-PCR analysis in combination with DNA sequencing confirmed that isoenzyme B transcripts obtained from X. laevis ovary and liver indeed harbor the codon for Thr223 (data not shown).

Expression of GnTI isoenzymes A and B in X. laevis tissues
Polyadenylated RNA was isolated from various X. laevis tissues and subjected to northern blotting analysis with a cDNA fragment corresponding to the complete coding region of isoenzyme A. This probe also hybridizes with transcripts encoding isoenzyme B. In all tissues examined, one major band of 3.0 kb was detected, suggesting that isoenzyme A and B mRNAs are of similar size. An additional 3.4-kb transcript appears to occur exclusively in the liver and may be the result of incomplete RNA maturation or transcription from alternative transcription initiation sites. Expression was highest in liver, with low levels of GnTI mRNA being present in other tissues such as ovary, muscle and skin (Figure 3A).



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Fig. 3. Expression of GnTI isoenzymes A and B in X. laevis tissues. (A) Poly(A)+ -RNA (2 µg) isolated from X. laevis ovary (O), liver (L), muscle (M), and skin (S) was subjected to northern blotting analysis as described in Materials and methods. The membrane was hybridized with a 32P-labeled probe corresponding to the entire coding region of GnTI isoenzyme A. The migration positions of RNA standards are indicated. (B) Total RNA (0.5 µg) isolated from X. laevis heart (H), ovary (O), liver (L), muscle (M), and kidney (K) was subjected to RT-PCR as outlined in Materials and methods. The samples were exhaustively treated with the restriction enzyme RsaI prior to analysis by Southern blotting with a 32P-labeled probe corresponding to the entire coding region of GnTI isoenzyme A. The amplified isoenzyme A sequence contains a diagnostic RsaI site that is not present in the respective isoenzyme B fragment. The bands corresponding to isoenzyme A (399 bp and 556 bp) and isoenzyme B (955 bp) transcripts are indicated.

 
RT-PCR analysis of total RNA was used to distinguish between isoenzyme A and isoenzyme B mRNAs, exploiting a diagnostic restriction site within the coding region of isoenzyme A. Both isoenzyme A and isoenzyme B transcripts were detected in all tissues examined, albeit in different amounts and ratios, thus suggesting that both genes are ubiquitously expressed (Figure 3B).

The genome of X. laevis harbors two closely related GnTI genes
The occurrence of both isoenzyme A and isoenzyme B mRNAs in most tissues suggests the existence of two transcriptionally active GnTI genes in the genome of X. laevis. To test this hypothesis, genomic DNA was isolated and subjected to Southern blotting analysis with a cDNA probe comprising the complete coding region of isoenzyme A. Under high-stringency conditions, two discrete bands were observed on digestion of the DNA with the restriction enzymes HindIII and EcoRI, whereas XhoI gave rise to a diffuse double band. Treatment with PstI yielded one major band with an intensity about twice as high as that of the bands obtained with the other enzymes. This suggests that this band represents two fragments of the same size (Figure 4). Because none of the enzymes cleaves within the coding region of either isoenzyme A or isoenzyme B, we conclude that X. laevis GnTI isoenzymes A and B are encoded by two separate genes.



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Fig. 4. The genome of X. laevis harbors two closely related GnTI genes. Genomic X. laevis DNA (15 µg) was exhaustively digested with the restriction enzymes XhoI (X), HindIII (H), EcoRI (E), and PstI (P) prior to analysis by Southern blotting with a 32P-labeled probe corresponding to the entire coding region of GnTI isoenzyme A. The migration positions of DNA standards are indicated.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We have isolated and characterized cDNAs for two closely related forms of GnTI expressed in X. laevis tissues. The catalytic domains of both variants share pronounced sequence similarity with their counterparts of mammalian, worm, and plant origin. Both isoenzymes were found to exhibit enzymatic activity, but isoenzyme B is less efficiently synthesized in vivo and thus probably of lower physiological relevance than isoenzyme A. However, a single amino acid substitution (Thr223->Ala) was sufficient to provide isoenzyme B with features similar to isoenzyme A. Replacement of the corresponding residue of isoenzyme A (Ala227) with threonine gave rise to an enzymatically inactive protein. According to the recently determined 3D structure of the catalytic domain of rabbit GnTI (Ünligil et al., 2000Go), this amino acid is not positioned in the close proximity of the active site of the enzyme. This is consistent with our observation that presence of Thr223 does not directly compromise the catalytic capacity of isoenzyme B. However, the corresponding residue of rabbit GnTI (Ala226) is part of an {alpha}-helix involved in the linkage of the two subdomains of the enzyme. Substitution of Ala226 with threonine could interfere with the formation of this {alpha}-helix and thus with proper folding of the protein.

Two mammalian cell lines that lack endogenous GnTI activity were previously derived by means of their resistance to toxic plant lectins. In both cases, this phenotype is due to a single point mutation within the catalytic domain of the enzyme. Replacement of Gly320 with Asp accounts for the GnTI deficiency of baby hamster kidney RicR14 cells (Opat et al., 1998Go). This substitution presumably prevents ordering of a loop involved in binding of acceptor substrates (Ünligil et al., 2000Go). Replacement of Cys123 with Arg is responsible for the fact that the GnTI protein synthesized by CHO Lec1 cells fails to exhibit enzymatic activity. It has been originally suggested that Cys123 may form part of the active site of GnTI (Puthalakath et al., 1996Go). However, the 3D structure of the enzyme indicates that this amino acid is not critical for the catalytic mechanism. Rather, it appears that the mutation of Cys123 into arginine destabilizes the enzyme because this residue points into the core of the protein (Ünligil et al., 2000Go).

The occurrence of multiple GnTI forms in an organism was also recently reported for C. elegans. The genome of this nematode contains three GnTI genes, all of them encoding functional enzymes. It was originally described that gly-12 and gly-14, but not gly-13, exhibit GnTI activity (Chen et al., 1999Go). However, recent studies revealed that gly-13 is also enzymatically active (S. Chen, A. Spence, and H. Schachter, personal communication).

Based on the crystal structure of rabbit GnTI, it was proposed that the catalytic domain starts with a ß strand beginning at residue 107 (Ünligil et al., 2000Go). Indeed, experimental evidence has been previously provided that 106 amino acids may be removed from the amino terminus of the enzyme without deleterious effects on its catalytic capacity. Any further truncation of the protein resulted in complete loss of enzymatic activity, probably due to destabilisation or misfolding (Sarkar et al., 1998Go). Soluble versions of both X. laevis GnTI isoenzymes A and B starting at the equivalent positions (Val108 and Val104, respectively) were found to be catalytically active, as already previously reported for N. tabacum GnTI (Strasser et al., 1999Go). These results are consistent with amino acid alignments that revealed that the first GnTI region displaying striking sequence similarity across species commences at this particular position. However, expression of an analogously truncated form of the putative Drosophila melanogaster GnTI orthologue in insect cells led to intracellular accumulation of an enzymatically inactive protein (J. Mucha et al., unpublished results).

No apparent sequence similarity was previously observed for the CTS regions of GnTI from mammals, worms, and plants. Similarly, the corresponding sequences of X. laevis GnTI isoenzymes A and B lack any striking similarity with their animal and plant counterparts. However, the CTS regions of both rabbit and N. tabacum GnTI are sufficient to mediate the retention of reporter proteins in the medial part of the Golgi apparatus, the proper subcellular location of these enzymes (Burke et al., 1992Go; Essl et al., 1999Go). Interestingly, the glycosylation defect of GnTI-deficient A. thaliana plants can be complemented by transformation with human GnTI cDNA (Gomez and Chrispeels, 1994Go). Functional expression of human GnTI was also achieved in Spodoptera frugiperda Sf9 cells, with the observed in vivo activity of the recombinant human enzyme suggesting a proper location in the medial-Golgi region (Wagner et al., 1996Go). Introduction of A. thaliana GnTI cDNA into CHO Lec1 cells restored the synthesis of complex-type N-glycans in the latter cells (Bakker et al., 1999Go). It was also reported that rat GnTI was correctly targeted to the Golgi apparatus of the yeast Saccharomyces cerevisiae (Yoshida et al., 1999Go). Despite their sequence diversity, the CTS regions of both animal and plant GnTI variants appear similarly effective in mediating the retention of exogenous proteins in the Golgi apparatus of various heterologous cell types, a feature that could be exploited to modify the glycosylation potential of biotechnologically relevant host organisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Adult X. laevis frogs were provided by Dr. H. Wiener (Institut für klinische Pharmakologie, Universität Wien). Oligonucleotide primers were synthesized by Dr. G. Himmler (Institut für Angewandte Mikrobiologie, Universität für Bodenkultur Wien). Taq DNA polymerase and DNA-modifying enzymes were purchased from Promega. Restriction enzymes and DNA standards ({lambda} DNA cleaved with HindIII and EcoRI) were from Roche. [{alpha}-32P]dCTP (3000 Ci/mmol), Hybond-C and Hybond-N membranes were obtained from Amersham Pharmacia Biotech. RNA standards (0.24–7.4-kb ladder) were purchased from Life Technologies. Prestained protein standards were from New England BioLabs. Purified recombinant S. japonicum GST was obtained from Pierce. UDP-[6-3H]GlcNAc (18.9 Ci/mmol) was from New England Nuclear. All other reagents were purchased from Sigma unless stated otherwise.

Preparation of genomic X. laevis DNA
Freshly excised X. laevis liver (0.5 g) was submersed in 5 ml 50 mM sodium tetraborate, pH 9.6, 150 mM NaCl, 100 mM EDTA, and disrupted with a Dounce homogenizer. The homogenate was centrifuged for 10 min at 3000 x g, the supernatant discarded, and the pellet resuspended in 5 ml of homogenization buffer. Sodium dodecyl sulfate (SDS) was then added to a final concentration of 2%. After incubation for 1 h, the sample was extracted for 16 h with 2.5 ml each of water-saturated phenol and chloroform, then centrifuged for 10 min at 3000 x g. Two volumes of ethanol were added to the aqueous phase, and the precipitated material was collected by centrifugation for 10 min at 3000 x g. The pellet was resuspended in 1 ml of 5 mM Tris–HCl, pH 8.0, 15 mM NaCl, 0.5 mM EDTA containing 20 µg/ml DNase-free RNase and then incubated for 30 min at 37°C. Subsequently, SDS and proteinase K were added to final concentrations of 0.5% and 0.5 mg/ml, respectively, prior to incubation for 2 h at 37°C. The reaction was stopped by extraction with phenol/chloroform, and the isolated DNA was recovered by ethanol precipitation as above. All steps were performed at 4°C unless stated otherwise.

Generation of a genomic X. laevis GnTI probe
Degenerate sense (5'-GTIGTIGA(A/G)GA(T/C)GA(T/C)(C/T) TIGA(A/G)G-3'; I = inosine) and antisense (5'-G(C/T)TG(A/G) TC(A/G)AA(A/G)AA(C/T)TGICC(A/G)TG-3') oligonucleotide primers were designed based on highly conserved regions of previously cloned GnTI cDNAs. Genomic X. laevis DNA (200 ng) was subjected to PCR with 0.5 unit Taq DNA polymerase and 100 pmol of each primer in 50 µl 50 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, and 125 µM each of dATP, dTTP, dCTP, and dGTP. The samples were first incubated for 4 min at 94°C, then amplification was carried out for 30 cycles at 94°C (40 s), 52°C (2 min), and 72°C (1 min). The final elongation step was extended to 3 min. The product (363 bp) was extracted from an agarose gel slice with the aid of QIAEX II (Qiagen) and subcloned into the plasmid pCR II (Invitrogen) using the TA Cloning kit (Invitrogen).

Isolation of mRNA from X. laevis tissues
Selected tissues were excised from adult X. laevis frogs and immediately frozen in liquid N2. Total RNA was isolated according to Chomczynski and Sacchi (1987)Go. Briefly, tissues (0.2 g) were thawed in 1 ml 25 mM sodium citrate, pH 7.0, 4 M guanidinium thiocyanate, 0.5% N-lauroyl sarcosine, and 0.1 M 2-mercaptoethanol and were disrupted with an Ultra-Turrax homogenizer. Then we added 0.1 ml of 2 M sodium acetate, pH 4.0, followed by 1 ml of water-saturated phenol and 0.2 ml of chloroform/isoamylalcohol. The samples were vigorously mixed and then centrifuged at 4°C for 10 min at 10,000 x g. One volume of isopropanol was added to the aqueous phase, followed by incubation for 1 h at –20°C. The precipitated RNA was then recovered by centrifugation as above.

Oligo(dT)-cellulose (150 mg) was activated according to the instructions of the manufacturer (Amersham Pharmacia Biotech), packed into a minicolumn and then equilibrated with 20 mM Tris/HCl, pH 7.6, 0.5 M NaCl, 1 mM EDTA containing 0.1% N-lauroyl sarcosine (binding buffer). Total RNA (0.3 mg) was resuspended in 50 µl 0.5% SDS and incubated for 5 min at 65°C prior to addition of 50 µl twofold concentrated binding buffer. The sample was then applied to the column, which was subsequently washed with 5 ml of binding buffer. Poly(A)+-RNA was then eluted with 2 ml of 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 0.05% SDS, and concentrated by ethanol precipitation.

Construction of an X. laevis ovary cDNA library
Double-stranded cDNA was synthesized from X. laevis ovary poly(A)+-RNA (5 µg) using the cDNA Synthesis kit (Amersham Pharmacia Biotech) according to the instructions of the manufacturer. The products were subcloned into the Lambda ZAP II vector (Stratagene) prior to in vitro packaging with Gigapack III Gold Packaging Extract (Stratagene). The cDNA library thus obtained consists of > 95% recombinant phages, with ~ 70% containing inserts larger than 500 bp.

Isolation of X. laevis GnTI cDNA clones
The genomic X. laevis GnTI probe (363 bp) was labeled with 100 µCi [{alpha}-32P]dCTP using the Oligolabelling kit (Amersham Pharmacia Biotech). The product was isolated using the QIAquick Nucleotide Removal kit (Qiagen). Approximately 5 x 105 plaque forming units of the X. laevis ovary cDNA library were blotted on Hybond-N membranes. Filters were hybridized with 106 c.p.m./ml of the radiolabeled probe in 0.5 M sodium phosphate buffer, pH 7.4, 7% SDS, 0.5 mM EDTA, for 16 h at 65°C, then washed in 2x sodium chloride–sodium citrate (SSC) (1x SSC is 0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 0.1% SDS at 60°C and finally in 0.5x SSC, 0.1% SDS at 60°C. Plaque-purified clones were converted into pBlueScript SK(–) phagemids by in vivo excision with the helper phage R408 (Stratagene) following the protocol of the supplier.

Southern blotting analysis
Genomic X. laevis DNA (15 µg) was exhaustively digested with the indicated restriction enzymes and then subjected to electrophoresis in a 0.8% agarose gel. The separated DNA fragments were transferred by capillary blotting under neutral conditions (20x SSC) onto a Hybond-N membrane. A cDNA probe corresponding to the complete coding region of X. laevis GnTI was isolated and labeled with [{alpha}-32P]dCTP essentially as above. The blot was hybridized with 106 c.p.m./ml of the 32P-labeled probe for 16 h at 65°C, then washed in 2x SSC, 0.1% SDS at 60°C and finally in 0.1x SSC, 0.1% SDS at 65°C.

Northern blotting analysis
Poly(A)+-RNA (2 µg) from selected X. laevis tissues was separated by electrophoresis in a 1.2% agarose gel in the presence of 2.2 M formaldehyde. Transfer onto a Hybond-N membrane was achieved by capillary blotting in 20x SSC. The blot was hybridized with 106 c.p.m./ml of 32P-labeled X. laevis GnTI cDNA for 16 h at 60°C, then washed in 2x SSC, 0.1% SDS at 60°C and finally in 0.5x SSC, 0.1% SDS at 60°C.

RT-PCR analysis
Total RNA (5 µg) from various X. laevis tissues was reverse transcribed using the First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech). cDNA corresponding to 0.5 µg total RNA was subjected to PCR for 35 cycles using X. laevis GnTI-specific sense (5'-CTCCACTCTTACCGTCC-3') and antisense (5'-GCCTACTTAACCATCAGCAG-3') primers and an annealing temperature of 49°C. Products were digested with the restriction enzyme RsaI prior to analysis by electrophoresis on a 1.5% agarose gel and Southern blotting with 32P-labeled X. laevis GnTI cDNA.

DNA sequencing
Sequences of subcloned X. laevis GnTI cDNAs were determined by the dideoxynucleotide chain termination method using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit and an ABI PRISM 310 Genetic analyser (Perkin-Elmer). Vector-derived or gene-specific oligonucleotides were used as primers. Analysis of DNA sequences was carried out using DNASTAR software. The BLAST program (Altschul et al., 1997Go) was used to search GenBank/EMBL sequence databases for homologous sequences.

Site-directed mutagenesis
The oligonucleotide 5'-GTATTTTCAGGCAACTATCCCCCTTCTGC-3' was used to introduce the substitutions Thr223->Ala and Ser226->Pro simultaneously into X. laevis GnTI isoenzyme B. This site-directed mutagenesis was performed using the Muta-Gene Phagemid in vitro Mutagenesis kit (Bio-Rad) according to the instructions of the manufacturer. The individual mutations Thr223->Ala and Ser226->Pro were created by overlap extension according to Ho et al. (1989)Go. The following overlapping primers were used: 5'-GGGAGATAGTTGCCTGAAAATACTCAT-3' and 5'-AGTATTTTCAGGCAACTATCTCCCTTCT-3' (Thr223->Ala), and 5'-TGCAGAAGGGGGATAGTTGTCT-3' and 5'-TCAGACAACTATCCCCCTTCTGCAAAAGGACCGT-3' (Ser226->Pro).

The replacement of Ala227 with Thr in X. laevis GnTI isoenzyme A was performed by inverse PCR. A cDNA fragment encoding the catalytic domain of the enzyme was generated by PCR with the primers 5'-GCCGCTGCAGATTCCAATACTAGTGGTGGC-3' (sense) and 5'-CCGGGGTACCGGAATTCGAGATCCATTTAGGTCCAC-3' (antisense) and ligated into pCR 2.1-TOPO (Invitrogen). Plasmid DNA (1 ng) isolated from a selected clone was subjected to inverse PCR with 3 units pfu DNA polymerase (Promega), 10 pmol each of the phosphorylated primers 5'-CAACTCTCCCCCTTCTACAAAAG-3' and 5'-TCTGAAAATACTCATAAAAATCTGGAG-3', and 0.5 mM each of dATP, dTTP, dCTP, and dGTP in 50 µl of the buffer supplied by the manufacturer of the enzyme. The sample was first incubated for 3 min at 94°C, then amplification was carried out for 40 cycles at 94°C (1 min), 55°C (1 min), and 72°C (6 min). The reaction products were ligated and transformed into competent bacteria. Positive clones were identified by DNA sequencing.

Construction of expression vectors
Fragments encoding the catalytic domains of X. laevis GnTI isoenzymes A and B were obtained by PCR with the primers 5'-CCGGATCCATTCCAATACTAGTGGTGGC-3' (sense) and 5'-CGGGATCCGAGATCCATTTAGGTCCAC-3' (antisense) and ligated into pCR II. The inserts (1.0 kb) of the resulting plasmids were transferred as BamHI fragments into the corresponding site of pAcSecG2T (PharMingen). In these constructs, the truncated X. laevis GnTI genes are placed downstream of the complete coding region of S. japonicum GST headed by the p67 leader sequence.

Alternatively, the regions corresponding to the catalytic domains of native and mutant isoenzymes A and B were amplified by PCR with the primers 5'-GCCGCTGCAGATTCCAATACTAGTGGTGGC-3' (sense) and 5'-CCGGGGTACCGGAATTCGAGATCCATTTAGGTCCAC-3' (antisense), digested with PstI and EcoRI and ligated into the corresponding sites of pVTBacHis-1 (Sarkar et al., 1998Go). In this plasmid, the truncated enzyme sequences are positioned behind a leader domain encoding the melittin signal peptide followed by a linker region containing six consecutive histidine residues and an enterokinase cleavage site.

The complete coding sequence of isoenzyme B cDNA was generated by PCR using sense (5'-ATAACTGCAGCGGATCCAGGGGAATGCCGCGC-3') and antisense (5'-CGGGATCCGAGATCCATTTAGGTCCAC-3') primers and subcloned into pCR II. The 1.4-kb insert was excised with BamHI and cloned into BamHI-digested pVL1393 baculovirus transfer vector (PharMingen).

In all baculovirus constructs used, the heterologous sequences are placed under control of the strong constitutive polyhedrin promoter.

Expression of X. laevis GnTI in insect cells
Insect cell lines (Sf9, Sf21) were grown in IPL-41 medium (Sigma) containing 5% heat-inactivated fetal bovine serum (Life Technologies). Each resulting recombinant transfer vector (1 µg) was cotransfected with 200 ng BaculoGold viral DNA (PharMingen) into Sf9 cells using Lipofectin (Life Technologies) as recommended by the manufacturer. After 5 days at 27°C, supernatants containing recombinant virus were used for infection of Sf21 cells. Cells and culture media were harvested after 4 days at 27°C and subjected to enzymatic analysis and immunoblotting.

Purification of recombinant X. laevis GnTI isoenzymes
Culture supernatants (50 ml) of Sf21 cells infected with the respective baculoviruses were cleared by low-speed centrifugation and then dialyzed against 2 x 2 L of 10 mM sodium phosphate buffer, pH 7.0, 40 mM NaCl, 0.02% NaN3. After addition of 20 mM imidazole, the retentate was centrifuged for 30 min at 20,000 x g. The resulting supernatant was loaded onto a 5 ml-column of Chelating Sepharose (Pharmacia) charged with Ni2+ ions, equilibrated in the same buffer. The matrix was washed successively with 40 and 80 mM imidazole prior to elution of the bound enzyme with 250 mM imidazole in dialysis buffer. After concentration by ultrafiltration, the eluate was analysed by SDS–PAGE and silver staining. The GnTI content of the sample was estimated by densitometric analysis of the stained gel, using bovine serum albumin as a standard.

Assay of GnTI activity
Cell pellets were resuspended at a concentration of 107 cells per ml of 0.2 M 2-(N-morpholino)ethane-sulfonic acid, pH 6.3, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 5 µg/ml leupeptin, 5 µg/ml E-64 (extraction buffer) and incubated for 30 min at 0°C. GnTI activity assays were performed in a total volume of 20 µl of half-concentrated extraction buffer containing 0.2 mM pyridylaminated Man3GlcNAc2, 2 mM UDP-GlcNAc, 5 mM AMP, and 20 mM MnCl2 at 37°C. Swainsonine (5 µg/ml) and 2-acetamido-1,2-dideoxynojirimycin (0.1 mM; a kind gift of Prof. A. Stütz, Technische Universität Graz, Austria) were included to prevent degradation of the product by endogenous {alpha}-mannosidase II and ß-N-acetylglucosaminidases, respectively. Km(app) values were determined for pyridylaminated Man3GlcNAc2 (concentration range: 0.1–2 mM) using double-reciprocal plots. The samples were then analyzed by reversed-phase high-performance liquid chromatography as reported previously (Altmann et al., 1993Go).

Alternatively, GnTI activity was determined with 0.5 mM Man{alpha}1–6(Man{alpha}1–3)Manß-O-(CH2)7CH3 (Man3-octyl) and 0.1 mM UDP-[6-3H]GlcNAc (2000–3000 c.p.m./nmol) as substrates under otherwise the same conditions as above. Enzyme reactions were stopped by addition of 0.5 ml of 20 mM sodium tetraborate containing 2 mM EDTA. The radioactive product was isolated by anion-exchange chromatography and quantified by liquid scintillation counting as previously described (Altmann et al., 1993Go).

Western blotting analysis
Lysates and culture supernatants of infected Sf21 cells were subjected to 12% SDS–PAGE under reducing conditions. Fractionated proteins were electrophoretically transferred onto Hybond-C membranes. The blots were incubated with affinity-purified rabbit antibodies to S. japonicum GST (Santa Cruz Biotechnology) or a mouse monoclonal antibody to the enterokinase recognition sequence (Invitrogen). Detection of bound antibodies was achieved with goat anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch) using the chromogenic substrate 4-Cl-1-naphthol and H2O2 or Supersignal West Pico Chemiluminescent substrate (Pierce).

Other methods
Total cellular protein was determined by the Bradford method with the Bio-Rad Protein Assay kit (Bio-Rad), using bovine serum albumin as a standard. Densitometric analysis was done using ImageQuaNT v4.2 software (Molecular Dynamics).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are grateful to Dr. Christelle Breton (Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France) and Dr. Georg Casari (Lion Bioscience AG, Heidelberg, Germany) for assistance with protein secondary structure prediction. This project was funded by grants P10962-GEN and P14343-GEN from the Austrian Science Foundation.

The nucleotide sequences presented in this article have been deposited in the GenBank data base (accession numbers X83975, Y16819).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHO, Chinese hamster ovary; CTS, cytoplasmic-transmembrane-stem; ER, endoplasmic reticulum; GnTI, ß-1,2-N-acetylglucosaminyltransferase I; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT, reverse transcriptase; SDS, sodium dodecyl sulfate; SSC, sodium chloride–sodium citrate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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