Department of Molecular Biology, Unit of Fundamental and Applied Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, B-9000 Ghent, Belgium
Received on May 9, 2000; revised on October 5, 2000; accepted on October 14, 2000.
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Abstract |
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Key words: CMP-NeuAc/Glycoconjugate/Surface plasmon resonance/Sialyltransferase/ST6GalI
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Introduction |
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All cloned glycosyltransferases, including sialyltransferases, have the same type II membrane topology. A short cytoplasmic tail precedes a transmembrane domain, a proline-rich stem region, and a large carboxy-terminal domain responsible for the catalytic activity in the lumen of the Golgi apparatus (Paulson and Colley, 1989; Kitazume-Kawaguchi et al., 1999
). Apparently, only the latter domain is needed for full enzymatic activity. A soluble, active form of the protein can also be found in serum, resulting from the proteolytic cleavage in the stem region. Two isoforms of ST6GalI that differ in only one amino acid in the catalytic domain show different catalytic activity and are differently processed (Ma et al., 1997
)
In contrast to what can be expected from this common structure, little sequence homology is found among the different sialyltransferases. Three sequence motifs with high homology were described within the catalytic domain (Wen et al., 1992; Datta and Paulson, 1997
). They are called the long, the small, and the very small sialyl motifs. Degenerated primers based on the sequences of these motifs allowed the cloning of several new sialyltransferases (Tsuji, 1996
; Giordanengo et al., 1997
; Samyn-Petit et al., 2000
). The presence of these common motifs in all cloned eukaryotic sialyltransferases points at an essential role in the structure or specific activity of the enzyme. The involvement of certain residues within the long sialyl motif in donor binding has been clearly demonstrated (Datta and Paulson, 1995
). Since all sialyltransferases known so far use the same donor substrate, the existence of a common motif is not surprising. Based on mutation analysis, the small sialyl motif was related to both donor and acceptor binding (Datta et al., 1998
). Different sialyltransferase types use different acceptors. Therefore, a common motif involved in the binding with the acceptor substrate is not expected. In this latter study, researchers were unable to analyze one of their mutants with much lowered activity because of the lack of appropriate tools. Indeed, classical kinetic studies that determine Km values for the donor and acceptor substrates are only possible with enzymatically active proteins. Little is known about the amino acids essential for the specific activity of sialyltransferases or for the interaction with their substrates.
We generated a collection of mutant human ST6GalI to search for residues involved in acceptor binding. The frame for mutagenesis was the C-terminal part, including the small and the very small sialyl motifs, immediately following the long sialyl motif. This region was chosen as a first target because the only amino acids involved in acceptor binding were previously shown to be situated there. Because several mutants expressed very low or no activity, tools other than classical measurement of Km values were optimized. For the first time, surface plasmon resonance was successfully exploited in directly determining the binding of a glycosyltransferase to its acceptor substrate.
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Results |
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Because mass screenings were planned, transfection protocols for COS1 and HEK293 cell lines were optimized for their use in 96-well microplates. HEK293 cells were chosen, because they gave the best results as to yield and reproducible transfection efficiency. Moreover, no endogenous sialyltransferase activity could be demonstrated in the extracellular medium after transfection with blank vector.
Homology studies between different sialyltransferases revealed the sialyl motifs (Wen et al., 1992; Datta and Paulson, 1997
). Only in the small motif, amino acids involved in acceptor binding were described. Therefore we chose a window for random mutagenesis, including this region. In the final expression product, the C-terminal 139 amino acids, including the small (from P321 to F343) and the very small sialyl motifs (from G367 to V379), were potentially mutated. The introduction of mutations was done under conditions of error-prone polymerase chain reaction (PCR). High Taq DNA polymerase and Mg2+ concentrations and trace amounts of Mn2+ were responsible for both transversion and transition mutations. In the presence of 0.2 mM MnCl2, the best yield in single mutants was obtained. By exchanging the desired mutated PCR fragment in the expression vector containing wt ST6GalI, a mutant library was created. Each mutant vector was amplified separately after transformation of competent E. coli cells. Subsequently, each of them was transfected to HEK293 cells present in the wells of 96-well microplates.
The culture medium of two subsequent expression rounds was combined (400 µl total). To check the transfection efficiency, a dot-blot immunoassay was performed. The presence of expression product was confirmed using anti-E-tag antibody, a peroxidase-conjugated secondary antibody, and a luminescent substrate. Quantification of the signal was possible using LumiImager. Because the affinity tag is situated C-terminally, only full-length products are measured. In that way, mutants that introduced a stop codon or had impaired folding or secretion are eliminated from further investigation. The sialyltransferase activity in the culture medium was measured using a high through-put assay in 96-well microplates (Laroy et al., 1997). The ratio of both parameters allows us to determine the specific ST6GalI activity of the mutant and can be compared with that of wt enzyme. Mutants with lowered activity were isolated. Improved activity was not found. Sequencing of the mutated fragment revealed the mutations responsible for altered activity. Those mutants with a single mutation were selected for further investigation (Table I). Many reasons for the inactivating effect of the mutation can be expected. Affinity for both donor and acceptor substrate may be affected, the catalytic domain may be hit, or a conformational change may have occurred.
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Control of the affinity of wt ST6GalI with acceptor substrate using surface plasmon resonance
The most common way to analyze enzyme mutants is measuring apparent Km values. However, when no or very low remaining catalytic activity is left, this becomes impossible. The only possibility is to look at direct interaction. Surface plasmon resonance is the physical phenomenon used by BIAcore to measure such interactions (Fagerstam et al., 1991; Johnsson et al., 1991
). However, for the analysis of the interaction between enzymes and their substrates, this is not an obvious technique because of the fast on/off rate of binding. Theoretically, two setups are possible in BIAcore, depending on which component is immobilized on the sensor chip. At first sight, immobilization of the acceptor substrate was most obvious. In that way, both wt and mutant enzymes could be analyzed on the same sensor chip. As an acceptor we chose asialofetuin. The N-linked glycans on this protein were previously shown to be good acceptor substrates for ST6GalI (Mattox et al., 1992
; Laroy et al., 1997
). Using amine coupling, different amounts of this protein (ranging from 2000 to 10,000 response units) were covalently coupled to a CM5 sensor chip. As a negative control, a blank chip was used. After exposure of the sensor chip to culture medium containing wt ST6GalI, no specific binding event could be demonstrated. Different conditions were tried, such as 100-fold concentration of the culture medium, other buffer systems (100 mM HEPES pH 7.2; 100 mM sodium cacodylic acid pH 7.2; 20 mM sodium acetate pH 5 and 6; 20 mM Tris-HCl pH 8 and 9.5), addition of cytidine 5'-diphosphate (CDP), cytidine 5'-monophosphate (CMP), or organic solvents (acetonitrile, methanol, DMSO). Because all were unsuccessful, we dropped this option.
Alternatively, the proteins present in the partially purified culture medium were immobilized on a CM5 sensor chip after concentration and buffer change. As a control, equally treated culture medium of cells transfected with empty vector was used. Originally, exposure of a sensor chip with wt ST6GalI to different concentrations of asialofetuin was unsuccessful. In these first experiments, HBS (10 mM HEPES pH 7.2; 150 mM NaCl; 0.005% v/v Surfactant P20) was used during binding. However, a specific binding signal appeared only at NaCl concentrations below 25 mM and at asialofetuin concentrations above 400 µg/ml (Figure 1A). Consequently, all further binding assays were done using 0.5 to 1 mg/ml of asialofetuin in 100 mM HEPES (pH 7.2) without salt. The same buffer was used as running buffer. Obviously, regeneration of the sensor chip was easily obtained after injection of 10 µl 1 M NaCl. Under these conditions, binding properties of wt ST6GalI enzyme with asialofetuin were characterized in more detail. No difference in binding was seen in the presence of CMP or CDP. Thus, acceptor binding does not need prior binding of donor substrate. In the presence of CMP-NeuAc, the binding signal was lowered. No more binding was seen at a donor concentration of 10 mM (data not shown). When 2 mM of donor substrate was injected during dissociation, a higher dissociation rate was obtained (Figure 1B). These results prove that the signals obtained are specific for binding between immobilized ST6GalI and asialofetuin. Moreover, these experiments show that sialyltransferase is enzymatically active in the absence of NaCl, which is in agreement with previous results describing assays for this enzyme in saltless buffer (Weinstein et al., 1982a; Datta and Paulson, 1995
).
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Discussion |
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One family of glycosyltransferases is the sialyltransferase family. These enzymes catalyze the transfer of sialic acid from its activated donor substrate, CMP-NeuAc, to the nonreducing end of a growing sugar chain. So far, 15 different kinds of sialyltransferases have been cloned (Tsuji, 1999). They are classified according to the acceptor they sialylate and to the linkage made. For example, ST6GalI, the enzyme used in this study, transfers sialic acid from CMP-NeuAc to a Galß1,4GlcNAc sequence in an
2,6-linkage (Weinstein et al., 1982a
,b). This lactosamine disaccharide is typically found on many glycoproteins. One of them is asialofetuin, the desialylated form of fetuin (Takasaki and Kobata, 1986
). This glycoprotein was previously shown to be a good acceptor for this enzyme (Mattox et al., 1992
; Laroy et al., 1997
).
The enzyme sequence as deduced from the human cDNA sequence (Grundmann et al., 1990) also suggests the common glycosyltransferase structure. So one could suspect high sequence homology between all sialyltransferase members. However, the opposite is true. Three domains with elevated homology were characterized previously and tentatively called the long (from R183 to T228 in the human enzyme), the small (from P321 to F343), and the very small sialyl motifs (from G367 to V379)(Wen et al., 1992
; Tsuji, 1996
). It has been reported that the long motif is situated in the donor-binding domain and the small motif is located in both the donor- and acceptor-binding sites (Datta and Paulson, 1995
; Datta et al., 1998
). Preliminary results suggest that the very small sialyl motif is needed for catalytic activity (Tsuji, 1999
). For the amino acid residues outside these motifs, little is known. However, because the members of the sialyltransferase family use different acceptor sugars, the binding site for this substrate may well be located there. ST6GalI was chosen as a model to evaluate the structurefunction relationship. Randomly mutated enzymes were checked for their specific activity and their binding to donor and acceptor substrate. Specifically, the binding site for the acceptor substrate was of interest. Asialofetuin was used not only in the activity assay (Laroy et al., 1997
) but also in the binding experiments.
Only part of the enzyme was mutagenized for our study. We chose the C-terminal part, including the small and the very small sialyl motifs but not the long one. The study of the homologous motifs suggested that the acceptor-binding site was located there. Single mutants with impaired activity were selected for further investigation. All mutants could be partially purified by affinity chromatography, indicating that the donor-binding site was not affected. To investigate whether the mutation caused a change in acceptor binding, surface plasmon resonance was used. This method was chosen because the measurement of apparent Km values became impossible for those mutants with low residual activity. Moreover, in the previously mentioned study of the small sialyl motif (Datta et al., 1998), at least one mutant was excluded from kinetic analysis for this reason.
Actual measurement of the interaction of sialyltransferase with asialofetuin using BIAcore technology was only possible in one configuration. Mutant or wt enzyme had to be immobilized on the sensor chip. Subsequent injection of asialofetuin only revealed a clear binding signal when this was done in low or salt-free buffers and at high acceptor concentrations. The first aspect indicates highly hydrophylic interaction; the latter demonstrates low-affinity binding. The conditions used had the consequence that mass transport effects and aspecific binding of the injected asialofetuin to the chip could not be excluded, making it impossible to determine adequate kon and koff rates. A control experiment, in which equally treated proteins of expression medium without sialyltransferase are immobilized to the chip, is essential to correct for these effects.
Upon injection of asialofetuin in the presence of CMP-NeuAc, the overall binding levels dropped. In that case, bound asialofetuin is sialylated by immobilized enzyme and released immediately. Alternatively, CMP-NeuAc can be injected after applying the acceptor to the sensor surface, resulting in a higher dissociation rate. The same explanation as before can be given. Both experiments prove that the signals obtained after correction for aspecific binding are specific for the interaction between the enzyme and its acceptor substrate. Moreover, they show that the enzyme is still active after immobilization and under the salt-free conditions used.
For wt enzyme, no difference in binding to acceptor was seen in the presence of CMP or CDP. We conclude that acceptor binding does not require prior donor binding. In contrast, acceptor binding is not influenced by the presence of CDP, a potent inhibitor of CMP-NeuAc. This means that also those mutants with impaired donor binding could be analyzed using this method.
Comparing the sensorgrams of mutant enzymes with those of wt enzyme led to the characterization of different types of mutants (Figure 2). For some mutants (such as E314G and N319Y) the impaired activity could be explained by the lowered or disappeared capability to bind to the acceptor. However, this was not always the case. Mutants K297E and Y281H for example, have no residual activity and lowered residual activity, respectively, but bind asialofetuin as efficiently as wt enzyme. In the former, this binding is not affected by the presence of CMP-NeuAc as with the wt enzyme. This clearly indicates that the mutation inactivates the catalytic center of the molecule. This is the first time that such a mutant is shown. For some other mutants, such as I328N and F401L, the lowered activity was associated with higher binding properties. Probably the improved binding has a negative influence on the catalytic activity. Two mutants in which a tyrosine was changed to an asparagine (Y355N and Y365N) had almost equal binding characteristics in the absence of NaCl. However, this binding is more resistant to higher salt concentrations. Both mutants have impaired activity. Without a three-dimensional structure, a conclusive explanation for this phenomenon is difficult to give.
All characterized mutations were randomly distributed along the mutated region. Only some of them were located in the small or the very small sialyl motif. Four mutants were evaluated in the small motif (M325I, I328N, E342G, F343S). All of them had lowered sialyltransferase activity and had changed binding characteristics to acceptor substrate. This is in accordance with Paulson et al. (1977), who link this motif to acceptor binding; they also mention the mutation of the phenylanaline residue on position 343, viz. in rat ST6GalI at position 340 (Datta et al., 1998
). However, they could not analyze it because of the low residual activity. We now can demonstrate that this mutant has impaired acceptor binding.
In addition, mutants outside these motifs were analyzed. All types as mentioned above were shown. This demonstrates that neither for acceptor binding nor for catalytic activity are the sialyl motifs sufficient. For the former, this is not surprising because different sialyltransferase types use different acceptor molecules.
These results clearly demonstrate the benefits of using BIAcore technology in the analysis of sialyltransferase mutants. It has hereby become possible to study the binding of mutants with the wild type acceptor without needing any residual enzymatic activity. This is a major advantage as compared to the use of classical studies that calculate Km values. Not only a change in affinity for the acceptor could be demonstrated by this method, but also other parameters, such as catalytic activity, could be deduced. Further improvement of the technique applied could even lead to a more detailed kinetic analysis of wt or mutant sialyltransferase. Therefore, more purified enzyme would be needed. Also, the use of the E-tag to immobilize fixed amounts of enzyme could be useful. This technique may also be of use to evaluate other sugar-transferring enzymes and will especially be valuable for enzymes for which three-dimensional structural data are or will become available.
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Materials and methods |
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Random mutagenesis was achieved using error-prone PCR with the expression plasmid as a template. This method was adapted from a previously described protocol (Leung et al., 1989). The N-terminal primer 5'-GCTGTTACTGCCAGGACCCAG-3' hybridizes in the coding sequence of the interferon-
secretion signal, the C-terminal primer 5'-CAACAGTCTATGCGGCACGC-3' partially in the coding sequence of the E-tag and partially in the 3'-untranslated region. 2.5 units of Taq polymerase were used in a PCR reaction performed in 20 mM Tris-HCl pH 8.4, 50 mM NaCl and 7 mM MgCl2, and 30 pmoles of each primer. Addition of 0.2 mM MnCl2 increased the mutation frequency of the polymerase. The latter was determined as the concentration at which 01 point mutations per 100 nucleotides are introduced. The reaction conditions were: 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min (30 cycles). The amplified product was purified using agarose gel electrophoresis and QIAquick (Qiagen, Chatsworth, CA). In the original expression vector, the coding sequence between a unique KpnI restriction site (at nucleotide 809 of the original clone) and a unique MunI restriction site in the E-tag (total length of 420 nucleotides) was interchanged with a mutant PCR fragment, prepared after restriction digestion with the same enzymes. Transformation of competent E. coli cells with the ligation mixture allowed the isolation of each mutant separately and their amplification. Purification was achieved as previously described (Birnboim and Doly, 1979
) or with Qiagen tips (Qiagen). The mutations were characterized by dideoxy double-stranded sequencing (Sanger et al., 1977
). The upstream primer was 5'-CCTAATTGTATGGGACCCATCTG-3' just preceding the KpnI restriction site. The downstream primer was the same as the one used for error-prone PCR.
Expression of wt- and mutant-soluble ST6GalI
Expression of wt-soluble ST6GalI and separate mutants was obtained after transient transfection of HEK293 cells, using calcium phosphate precipitation (Chen and Okayama, 1987). For mass screening of sialyltransferase mutants, 10,000 cells were transfected in 96-well microplates using 0.1 µg of purified DNA. For surface plasmon resonance experiments, 10 µg of purified DNA was used to transfect 1.5 million cells in medium flasks. After washing the transfected cells, expression was allowed for 48 h in Dulbeccos minimal essential medium enriched with insulin, transferrin, and selenium. After harvesting, expression was allowed for another 72 h in fresh medium. Both were combined for further experiments.
Analysis of the residual specific activity of ST6GalI mutants
Transfection medium from 96-well microplates was checked for protein expression using dot-blot immunodetection. After immobilization of the proteins on a nitrocellulose membrane (Schleicher & Schuell, Dassel, FRG), incubation with anti-E-Tag antibody (Pharmacia Biotech) as a primary antibody and with peroxidase-conjugated anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) as a secondary antibody allowed luminescent detection with Renaissance luminescence reagent (Du Pont, Wilmington, DE). Quantification was possible using Lumi-Imager and its software (Roche Molecular Biochemicals, Basel, Switzerland). The activity of the expressed sialyltransferases was analyzed as described previously (Laroy et al., 1997). Briefly, asialofetuin is used as an acceptor for radiolabeled sialic acid. After precipitation of the reaction product on glass fiber filters present in 96-well filtration plates, excess donor is washed away. The amount of radioactivity left on the filter is a measure for sialyltransferase activity. Both parameters permit us to draw conclusions on the specific activity of the mutants and their comparison with wt enzyme.
Partial purification of expression medium
Transfection medium was concentrated tenfold using micro-concentrators (MWCO 10; Vivascience, Westford, MA). Concentrated medium (500 µl) was supplemented with an equal volume of buffer A (10 mM MES, pH 7.2, 25% glycerol) and applied on a 1-ml glycosyltransferase affinity gel-CDP (Calbiochem-Novabiochem International, San Diego, CA), pre-equilibrated with buffer A. After washing the column with 15 ml buffer A, elution was started using 15 ml buffer B (10 mM MES, pH 7.2, 1 M NaCl, 25% glycerol). All steps were done at a flow rate of 0.2 ml/min. Fractions of 1.5 ml were collected and checked for the presence of E-tag using the dot-blot protocol mentioned above. Fractions containing the E-tag were combined.
Surface plasmon resonance
The interaction of asialofetuin (Sigma Chemical Co.) with immobilized wt and mutant sialyltransferases was analyzed by surface plasmon resonance using BIAcore 2000 and evaluated with BIAevaluation 2.1 software (Biacore, Uppsala, Sweden). The basic principles were reviewed previously (Fagerstam et al., 1991; Johnsson et al., 1991
). Two setups were worked out. By using amine coupling, asialofetuin (40 µg/ml in sodium acetate, pH 4) was immobilized on a CM5 sensor chip (research grade; Biacore) as described previously (Hutchinson, 1994
). Amounts ranging from 2000 to 10,000 response units were coupled. As a ligand, culture medium containing wt ST6GalI was injected under different conditions. Alternatively, proteins obtained after partial purification of the culture medium were coupled to the carboxy-methylated dextran matrix present on a CM5 sensor chip (research grade; Biacore) using amine coupling and following the manufacturers recommendations. Just before immobilization, eluted fractions containing the E-tag were combined. Glycerol was removed, and buffer was brought to 10 mM sodium acetate pH 5 using micro-concentrators (MWCO 10; Vivascience). Dot-blot immunodetection of the E-tag was used to measure the enzyme concentration. Equal amounts of E-tagged protein (i.e., wt or mutated enzyme) were applied to the activated sensor chip. All binding experiments were performed with asialofetuin (5001000 µg/ml) in 100 mM HEPES, pH 7.2, except where differently mentioned. Per injection, 20 µl was used. Dissociation occurred in 100 mM HEPES, pH 7.2, or in the same buffer with 150 mM NaCl. The flow rate was 10 µl/min. Dissociation experiments were carried out with 2 mM CMP-NeuAc (Sigma Chemical Co.) in 100 mM HEPES pH 7.2. Regeneration of the sensor chip was possible with 10 µl 1 M NaCl.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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