2Genetics Group, Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec H4P 2R2, Canada, 3Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B2, Canada, 4Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA, 5Laboratoire dIngénierie des Macromolécules, Institut de Biologie Structurale J.-P. Ebel, CNRS, Grenoble, France, and 6Instituto de Investigaciones Bioquimicas Fundacion Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina
Received on September 2, 1999; revised on November 8, 1999; accepted on November 8, 1999.
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Abstract |
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Key words: glucosyltransferase/baculovirus/acid phosphatase/folding/quality control
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Introduction |
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UGGT has been previously purified to homogeneity from rat liver and shown to have an apparent molecular weight of about 150 kDa on SDSpolyacrylamide gels (SDSPAGE) and 270 kDa under native conditions (Trombetta and Parodi, 1992). This enzyme catalyzes the transfer of a single glucose residue from UDP-glucose onto the distal mannose residue of the longest branch of the core oligosaccharide in an
-1,3 linkage (Trombetta et al., 1989
). Glycoproteins containing Man9GlcNAc2 glycans are better acceptors for glucose transfer than Man8GlcNAc2 which in turn are better than Man7GlcNAc2 (Sousa et al., 1992
). Oligosaccharides with a lower mannose content are not substrates for UGGT. Most importantly, the glucosylation reaction is far more efficient if the glycoprotein substrate is denatured. The effect of denaturation is believed to make polypeptide determinants required for recognition by UGGT accessible and UGGT may have an affinity for hydrophobic peptides (Sousa et al., 1992
; Sousa and Parodi, 1995
).
cDNAs encoding proteins with UGGT activity have been cloned from Drosophila melanogaster (GenBank Accession Number U20554; Parker et al., 1995) and from the fission yeast Schizosaccharomyces pombe, gpt1+ (GenBank Accession Number U38417; Fernández et al., 1996
). The sequence of the gene encoding a UGGT from Caenorhabditis elegans is also available (GenBank Accession Number U28735; Wilson et al., 1994
). These cDNAs all encode proteins of about 1500 amino acids with a N-terminal signal sequence and a C-terminal HDEL-type ER retention signal. The gene for UGGT is not essential in S.pombe and the gene disruption phenotype is only apparent under stress conditions (Fanchiotti et al., 1998
). The KRE5 gene of S.cerevisiae, which has the closest homology to UGGT, is essential for growth (Meaden et al., 1990
), and although there is no apparent UGGT activity in ER isolated from this organism, KRE5 mutations lead to alterations in the cell wall (Meaden et al., 1990
; Parodi, 1998
; Taylor, unpublished observations).
One of the major hurdles to the study of the enzymology of UGGT is the availability of Man9GlcNAc2 glycoprotein substrates. High-mannose oligosaccharides are transient species in the ER, they become substantially modified and remodelled as they proceed through the secretory pathway. Consequently, most secreted glycoproteins do not contain Man7-9GlcNAc2 acceptor glycans for UGGT and if they do, these oligosaccharides constitute only a small fraction of the total, as it is the case for ribonuclease B (RNase B) (Rudd et al., 1994; Zapun et al., 1997
). The mechanism for the transfer of N-linked oligosaccharides to proteins is conserved in vertebrates and yeast. However in yeast, the terminal mannose residue of the Man9GlcNAc2 core oligosaccharide remaining after glucose trimming by the ER glucosidases is removed by the action of an ER
-mannosidase which is the product of the gene MNS1 (Jelinek-Kelly and Herscovics, 1988
). The remaining terminal mannose units are the acceptors for the addition of further mannose residues by a Golgi
-1,3-mannosyltransferase encoded by the gene MNN1 (Graham et al., 1992
). Finally, one of the GlcNAc residues is also the site of attachment of an additional mannose residue by an
-1,6-mannosyltransferase encoded by the OCH1 gene (Nakayama et al., 1992
). Long polysaccharide chains are then built on these additional mannose residues to produce the typical heterogeneously hyperglycosylated yeast proteins. We have constructed a strain lacking these three genes (mns1, mnn1, and och1) to produce glycoproteins with Man9GlcNAc2 oligosaccharides. As the pathway of oligosaccharide biosynthesis in S.cerevisiae is well characterized and that not all genes are essential for growth, this organism can serve as a source of specific protein glycoforms.
Here, we describe the cloning of a cDNA from rat liver for the first mammalian UGGT (RUGT), its expression and the purification of active recombinant enzyme from insect cells using a baculovirus system. We present the kinetics of the purified enzyme and identify residues involved in catalytic activity. We also describe the development of the DT111 strain of S.cerevisiae which secretes Man9GlcNAc2 glycoproteins as a source of homogeneous substrates for in vitro glucosylation.
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Results |
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Activity of RUGT
The activity of the purified recombinant RUGT was tested with various glycoprotein substrates. As previously reported with the enzyme purified from rat liver (Trombetta and Parodi, 1992; Zapun et al., 1997
), the recombinant glucosyltransferase showed a greater activity towards reduced and unfolded RNase B than towards the native protein. Similarly, heat-treated SBA and urea-denatured Tg were better substrates than the native proteins (Figure 5A). These results demonstrate that the recombinant RUGT has the same preference for unfolded glycoproteins as the enzyme purified from rat liver tissue. On a molar basis, G0-AcP purified from DT111 incorporated 3H-glucose 130 times better than RNase B, 100 times more than SBA and four times more than Tg (Figure 5A). When UDP-glucose was used in large excess (labeled plus unlabeled) compared to the acceptor substrate and the reaction was allowed to proceed to completion, calculation of the molar incorporation of 3H-glucose showed that less than one glycosylation site of acid phosphatase gets glucosylated by RUGT. Thus, it appears that very few N-linked glycans present in G0-AcP are targets for glucosylation by RUGT (data not shown). Complete denaturation of G0-AcP by reduction of its disulfide bonds in the presence of 6 M guanidinium hydrochloride followed by desalting prior to glucosylation did not result in a greater incorporation of 3H-glucose (Figure 5A), indicating that a simple incubation at neutral pH is enough to denature G0-AcP to an extent sufficient for recognition by RUGT.
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Localization of the active site
The four residues conserved in most glycosyltransferases listed in Figure 2 were independently mutated to alanines in RUGT and the activity of the purified mutant enzymes compared to that of the wild-type enzyme using purified G0-AcP as a substrate. As shown in Figure 6A, the D1334A and D1336A mutants showed no detectable incorporation of 3H-glucose in G0-AcP. The Q1429A mutant showed a greater than 98% reduction in enzyme activity whereas the N1433A mutant showed about 15% of the activity of the wild-type enzyme (Figure 6A). The N-terminus of the C-terminal 37kDa band from RUGT (see Figure 3B, lane 7) was determined to be KTEEVKQDKDD which corresponds to residues 12201230 of RUGT. Because the results obtained with the RUGT mutants locate the active site of the enzyme to the C-terminus of the protein, we were interested to see whether the 37 kDa fragment produced by partial proteolysis retained activity. Therefore, the cDNA coding for this 37 kDa fragment was fused to the melittin signal peptide in the same way that we expressed the full-length RUGT and was introduced in baculovirus. This C-terminal 37 kDa fragment of RUGT was expressed and secreted from insect cells and Ni2+-purified fractions were shown to retain about 5% of the glucosyltransferase activity compared with the full-length RUGT clearly indicating that the active site lies within this region of the enzyme (Figure 6A). The autoradiogram was overexposed to show the weaker labeling of G0-AcP by the mutant RUGT enzymes compared with the wild-type. Similar yields of all four RUGT mutant proteins (D1334A, D1336A, Q1429A, and N1433A) were obtained from insect cells indicating that these point mutations did not substantially alter the stability of the enzyme.
Photoaffinity probes for nucleotide diphosphate sugar-utilizing enzymes have been developed and have proven very useful in the mapping of the active site of various enzymes (Drake et al., 1989; Battaglia et al., 1997
). More specifically, the photoaffinity probe [ß-32P]5N3UDP-glucose was used previously for the labeling of rat liver microsomal UDP-glucosyltransferases (Radominska and Drake, 1994
). Purified fractions of wild-type and mutant RUGTs were incubated in the presence of the photoaffinity probe and irradiated before being analyzed by SDSPAGE using rat liver microsomal fractions as a control. The results in Figure 6B clearly show that the probe bound to the full-length wild-type RUGT as well as to the Q1429A and N1433A mutants whereas the D1334A and D1336A mutant enzymes which failed to show any enzymatic activity, also failed to bind the probe. Interestingly, the probe also bound to the C-terminal 37 kDa fragment of the enzyme in the partially hydrolyzed preparation of RUGT. The other fragments of RUGT mapping to the C-terminus of the protein based on the Western blot of Figure 3B (lane 8) also bound the probe in the partially hydrolyzed preparation of RUGT (weakly labeled bands at ~150 kDa and ~92 kDa). Preincubation of the enzyme preparations with a 3-fold excess of unlabeled UDP-glucose reduced the photoincorporation of the probe into RUGT showing competition of the probe and the nucleotide sugar for the same binding site.
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Discussion |
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A classification of known glycosyltransferases has been proposed based on sequence homologies (Campbell et al., 1997). The various UGGTs were grouped together with the S.cerevisiae KRE5 protein in a small family (family 24). The sequence alignment of the C-terminal catalytic domain of the UGGTs with other glycosyltransferases (Figure 2) allows the merging of UGGTs with the glycosyltransferases of family 8. All the enzymes of this latter family transfer a monosaccharide unit from a nucleoside diphosphate donor to a specific substrate while retaining the
-anomeric configuration of the sugar ring (Campbell et al., 1997
). A single nucleophilic attack on the C1 carbon atom of the sugar would result in the inversion of the anomerisation. It has been speculated that the mechanism of the glycosyltransferases which conserve the anomeric configuration may require the formation of a covalent glycosyl-enzyme intermediate, which is then the target of a second nucleophilic attack by the substrate. Such a mechanism would require two acidic amino acids, one to carry out the first nucleophilic attack and form the glycosyl-enzyme intermediate, the second to act as a base to deprotonate the substrate to allow the second nucleophilic attack. Thus it is interesting in this respect to note that two of the four highly conserved residues in the enlarged family 24 of glycosyltransferases are aspartic acids. The essential role of these two residues in RUGT (D1334 and D1336) for the catalysis is clearly demonstrated by the total loss of activity and absence of photoaffinity labeling following their replacement with alanines. These results provide one more piece of experimental evidence to support the direct implication of the DXD motif in the catalytic activity of glycosyltransferases. Site-directed mutagenesis studies have shown the essential requirement of the aspartic acid residues in the DXD motif for catalytic activity of the yeast
-1,3-mannosyltransferase Mnn1p (Wiggins and Munro, 1998
) and the glucosyltransferase activity of the clostridial cytotoxin of Clostridium sordellii (Busch et al., 1998
). The replacement by alanines of the two other highly conserved residues in RUGT (Q1429 and N1433) resulted in enzymes with drastically reduced activities. It is possible that these latter residues are important for the binding of the sugar donor or to provide an adequate environment for the catalytic function of the two conserved aspartic acids.
In contrast to other eucaryotes, no UGGT-like activity has been found so far in the yeast S.cerevisiae (Fernández et al., 1994; Jakob et al., 1998
; Taylor, unpublished observations). The product of the gene KRE5 shows significant homology to RUGT over the entire protein with 20% identity. Despite an even higher level of sequence identity in the C-terminal 400 residues, the KRE5 protein lacks the two aspartic acids fully conserved amongst the glycosyltransferases of family 8 (Figure 2). The KRE5 protein confers resistance to the killer toxin (Meaden et al., 1990
) and is involved in the synthesis of cell wall ß-16 glucans. If the KRE5 protein were the ß-16 glucan synthetase using a nucleoside diphosphate as monosaccharide donor, the reaction catalyzed would inverse the anomeric configuration of the sugar ring and proceed via a mechanism totally different from the reactions catalyzed by the family 8 of glycosyltransferases, which would be consistent with the absence of the critical aspartic residues. Whether the protein encoded by KRE5 is a yeast UGGT with a different mechanism or whether it has evolved from a common ancestor and now carries out a totally different function is not clear, as the enzymatic activity of the KRE5 gene product remains unknown.
Our observations with G0-AcP indicate that RUGT glucosylates only very few of the N-linked glycans on the acceptor substrate. It is reasonable to assume that each glycoprotein substrate may have prefered sites of glucosylation. Mass spectrometry analysis following partial proteolysis (Taylor et al., unpublished observations) suggests that the sequence and/or the topology around the glycosylation sites occupied in G0-AcP is an important factor in recognition as some sites consistently appear to be preferentially glucosylated. This differential glucosylation model would imply the existence of strategic sites for the binding of calnexin/calreticulin in the mechanism of retention and refolding of glycoproteins in the ER.
The initial discovery and characterization of calnexin implicated it as a classical molecular chaperone for glycoproteins (Ou et al., 1993) which recognized the conformational state of a target protein. However, experiments with purified calnexin in vitro and monoglucosylated RNase B have conclusively shown that calnexin binds monoglucosylated RNase B irrespective of its folded state (Zapun et al., 1997
). Thus, the only component so far of the "calnexin cycle" which does recognize the conformation of proteins is UGGT and the question of how it performs this and other functions in concert with the other components of the cycle is of continuing interest.
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Materials and methods |
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For expression, the gene was cloned in the RsrII and KpnI sites of pFastBac1 with the following modifications. At the 5' end of the gene, the sequence coding for the first 18 amino acids of RUGT was replaced by a sequence coding for the 21 amino acids of the honeybee melittin signal peptide to facilitate the secretion of the protein from baculovirus-infected insect cells (Tessier et al., 1991). At the 3' terminus of the cDNA, the sequence coding for the ER localization signal HEEL was replaced by a sequence coding for a terminal (His)6 tag to facilitate the purification of the protein. For the expression of the C-terminal 37kDa fragment of RUGT, the amino acid sequence starting at residue 1220 of RUGT (KTEEVKQDKDD...) was fused to the melittin signal peptide as above. Four independent RUGT mutants D1334A, D1336A, Q1429A and N1433A were designed, amplified by PCR, cloned, and resequenced to confirm the presence of the mutations and the diagnostic PstI, BsrBI, XbaI, and NaeI sites respectively.
Baculovirus expression and purification from insect cells
RUGT constructs in pFastBac1 were handled as described by the manufacturer (Gibco/Life Technologies Inc.) to produce stocks of recombinant RUGT baculovirus. Sf9 insect cells were grown for 34 days in Spinner flasks in Sf900 II SFM serum-free medium (Gibco/Life Technologies Inc.) to a density of 23 x 106 cells/ml and infected with 1/20 of the culture volume of a recombinant RUGT baculovirus stock representing an M.O.I. of ~25 pfu/cell. The infected cells were then incubated at 27°C for up to 3 days. Cells were removed by centrifugation and medium adjusted to 40mM TrisHCl pH 7.5, 0.5M NaCl. The medium was concentrated ~20-fold at 4°C on YM30 membranes (Amicon) in the presence of 2 µg/ml of each of the protease inhibitors aprotinin, leupeptin, pepstatin A and E-64 (Boehringer Mannheim Canada). Two buffer exchanges were performed using Buffer ATC/PI (40 mM TrisHCl pH 7.5, 0.5 M NaCl, 5 mM CaCl2 with protease inhibitors). The concentrated/diafiltrated solution was centrifuged at 3000 x g for 10 min to remove some precipitated proteins and 10mM imidazole was added to the supernatant before loading twice by gravity flow onto a 1 ml Ni2+-NTA Superflow column (Qiagen Inc.) equilibrated in Buffer ATC/PI + 10 mM imidazole. The column was then washed by gravity with 20 column volumes of the same buffer. The elution step was carried out on a BioCAD Perfusion Chromatography Workstation (PerSeptive Biosystems, Inc.). The column was washed further with 10 column volumes of the same buffer at 2ml/min and proteins eluted with 20 column volumes of Buffer ATC/PI + 200mM imidazole at the same flow rate. Fractions were collected and analyzed by SDSPAGE and Western blot. The mutant forms of RUGT and the truncated 37kDa enzyme were also purified on a Ni2+-NTA column.
RUGT-containing fractions from the Ni2+-NTA column were diluted 10 fold with HQ buffer (40 mM TrisHCl pH 7.5, 2 mM CaCl2) and applied onto a 1 ml POROS 20HQ column (PerSeptive Biosystem, Inc.) for further purification. The protein was eluted at 1ml/min with a gradient of 0750 mM NaCl in HQ buffer over 30 column volumes and RUGT activity eluted at approximately 350 mM NaCl. The purity of the fractions was assessed by SDSPAGE and protein concentration was determined using the Bradford assay (Bio-Rad).
Production of acid phosphatase
The S.cerevisiae yeast strains used in this study were: W3031A (MATa, ade2, his3, leu2, trp1, ura3, can1) (Parlati et al., 1995), YNS-7A (MAT a, och1::LEU2, mnn1, his1, his3, ura3; generous gift from Y.Jigami) (Nakayama et al., 1992
) and DT111 (MAT a, och1::LEU2, mnn1, mns1, his1, his3, ura3). DT111 was constructed by disrupting the MNS1 gene of YNS-7A using an mns1::URA3 cassette (Camirand et al., 1991
) from plasmid pBHE5 (a generous gift from A.Herscovics). The URA3 gene was later deleted by selecting for 5-FOA-resistant colonies (Rose et al., 1990
). Endogenous acid phosphatase was produced from DT111 which secretes glycoproteins with N-linked Man9GlcNAc2 glycans. DT111 was grown in YPD medium supplemented with 150 mM KCl as an osmotic stabilizer at 30°C to an O.D.600nm of 5. In order to induce the expression of endogenous acid phosphatase, the inoculum was diluted 50-fold in SD-Pi medium (SD medium without inorganic phosphate) (Rose et al., 1990
) and supplemented with 150mM KCl and 2% glucose and grown at 30°C to an O.D. 600nm of 2. Acid phosphatase (G0-AcP) was purified from the culture medium by 50100 fold concentration/diafiltration against 10mM sodium acetate pH 5.0 followed by anion exchange chromatography on a 1 ml POROS 20HQ column in 20mM sodium acetate pH 5.0 on a BioCAD. Proteins were eluted at 1 ml/min over 30 column volumes using a linear gradient of 0600 mM NaCl. G0-AcP eluted at ~100 mM NaCl. Fractions were collected and aliquots were analyzed by SDSPAGE to confirm the presence of the protein. G0-AcP activity was monitored at O.D.405nm by the hydrolysis of 0.5 mg/ml p-nitrophenyl phosphate (pNPP; Sigma 104) at 37°C for 30 min in 25 mM sodium acetate pH 4.0. The reaction was terminated by adjusting the pH to ~9.5 with an equal volume of saturated Na2CO3.
Glucosyltransferase assay and kinetic measurements
G0-AcP was the major substrate glycoprotein used to determine RUGT activity in this study. Unfolded RNase B (Sigma) was prepared as described elsewhere (Zapun et al., 1997). Soybean agglutinin (SBA, Sigma) was denatured by heating at 100°C for 15 min. Bovine thyroglobulin (Tg; Sigma) was denatured in 8M urea and dialyzed against ddH2O. The G0-AcP acceptor-substrate (~1 µg) was mixed with RUGT in a 20 µl mixture containing TC buffer (20 mM TrisHCl pH 7.5, 10 mM CaCl2) and 5 µM uridine diphospho-D-[6-3H]-glucose (620Ci/mmol; Amersham Life Science Inc.) and incubated at 37°C for 12 h. Reactions were either analyzed by SDSPAGE followed by fluorography using Amplify (Amersham Life Science Inc.) or precipitated with ice-cold 10% trichloroacetic acid (TCA) in the presence of 500 µg of BSA, washed twice with TCA and the pellets resuspended in 50 µl 1M TrisHCl pH 8.0 before counting in a scintillation counter to quantitate the incorporation of 3H-glucose in acid phosphatase. For the determination of the Km of UDP-glucose, 50 nM RUGT was mixed with 0.5 µM of purified and desalted G0-AcP and 5800 µM UDP-glucose (mixture of labeled and unlabeled) in TC buffer. For the determination of the Km for G0-AcP, 50 nM RUGT was mixed with 5 µM UDP-[3H]glucose and 0.14 µM of purified and desalted G0-AcP in TC buffer. Glucosylation reactions were incubated at 37°C and aliquots of 20 µl were TCA precipitated at various times up to 120 min to determine the rate of incorporation of 3H-glucose in acid phosphatase.
Photoaffinity labeling
[ß-32P]5N3UDP-glucose (final concentration 40 µM; 25 Ci/mmol) was mixed with various fractions of RUGT and/or mutants for 1 min prior to UV-irradiation with a handheld lamp for 90 s at room temperature (UVP-11, 254 nm, Ultraviolet Products, Inc. San Gabriel, CA). To show competition for active site binding, a 3-fold excess of unlabeled UDP-glucose was preincubated for 15 min with the enzyme prior to addition of the labeled probe. Rat liver microsomes were used as controls and were prepared as described previously (Radominska and Drake, 1994). The labeling reactions were terminated as described previously and the samples processed for SDSPAGE, Coomassie blue staining/destaining and drying followed by autoradiography (Radominska and Drake, 1994
).
Production of polyclonal antibodies directed against the C-terminus of RUGT and N-terminal sequencing
A 15-mer peptide EEKELGTLHEEETQE (amino acid residues 15051519) was synthesized on an 8-branch MAP core (Applied Biosystems) using Fmoc chemistry and desalted on a C18 Sep-Pak reverse phase chromatography cartridge (Millipore/Waters). Rabbits were immunized as described previously (Cooper and Paterson, 1995). The resulting anti-RUGT-peptide serum also showed cross-reactivity to two insect cell proteins of about 62 and 70 kDa, respectively (Figure 3A). N-Terminal sequencing was performed with an Applied Biosystems model 470A gas-phase protein sequencer equipped with an on-line model 120A PTH amino acid separation system (Hewick et al., 1981
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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