Histidine 271 has a functional role in pig {alpha}-1,3galactosyltransferase enzyme activity

Brooke D. Lazarus, Julie Milland, Paul A. Ramsland, Effie Mouhtouris and Mauro S. Sandrin1

John Connell Laboratory for Glycobiology, The Austin Research Institute, Austin and Repatriation Medical Centre, Studley Road, Heidelberg 3084, Australia

Received on January 23, 2002; revised on July 2, 2002; accepted on July 25, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Gal{alpha}(1,3)Gal. This glycosyltransferase has been studied extensively because the Gal{alpha}(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Gal{alpha}(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in {alpha}(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.

Key words: {alpha}(1,3)galactosyltransferase/catalytic function/molecular modeling/UDP-galactose/UDP-N-acetylgalactosamine


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycosyltransferases are key enzymes involved in the synthesis of complex carbohydrates (Field and Wainwright, 1995Go). Each glycosyltransferase transfers a single monosaccharide unit to an acceptor molecule, usually an oligosaccharide that is coupled to either a protein or lipid (Joziasse, 1992Go). Glycosyltransferases that add a terminal sugar moiety to their specific acceptor molecule have received the most attention, because their products are usually involved in recognition and signaling events. However, the molecular basis of substrate specificity is uncharacterized and remains unclear.

The {alpha}-1,3galactosyltransferase (GT) enzyme catalyzes the transfer of a terminal galactose (Gal) moiety from the UDP-galactose (UDP-Gal) substrate to its acceptor molecule, N-acetyllactosamine (NAcLac) (Blanken and Van den Eijnden, 1985Go). This reaction forms the Gal{alpha}(1,3)Gal epitope, which has been shown to be the major xenoantigen in pig-to-human transplantation (Sandrin and McKenzie, 1994Go; Sandrin et al., 1993Go). The aim of this study was to examine the mechanism of GT catalysis and the interaction of this enzyme with the donor sugar substrate. GT is a member of the {alpha}-1,3Gal/GalNAc transferase family (family 6; http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html), which includes A transferase (Seto et al., 2000Go), B transferase (Orntoft et al., 1988Go), Forssman synthetase (Haslam and Baenziger, 1996Go), iGb3 synthase (Keusch et al., 2000Go), and GalNAcT-like transferase (accession no. AAF74758.1). Amino acid residues that could be used as potential targets for mutagenesis were identified by comparison of amino acid sequences of the family 6 members, previously reported mutagenesis of the human A and B transferases (Seto et al., 1997Go, 1999; Yamamoto and McNeill, 1996Go), chemical modification studies of GT (Stults et al., 1999Go), and the original published crystal structures of bovine GT (Boix et al., 2001Go; Gastinel et al., 2001Go).

The human A and B transferases have been studied extensively for amino acid–based structure/function because these two glycosyltransferases differ by only four amino acids (Yamamoto et al., 1990Go) yet catalyze the transfer of different terminal sugars. Human A transferase catalyzes the transfer of GalNAc from the donor molecule UDP-GalNAc to the precursor acceptor molecule H-substance (Fuc{alpha}1,2Galß1,3/4-R). In contrast, human B transferase catalyzes the transfer of Gal from the donor molecule UDP-Gal, to the same acceptor molecule (Yamamoto and Hakomori, 1990Go). A number of studies have systematically mutated these four amino acids (Arg176, Gly235, Leu266, and Gly268 in the A transferase; Gly176, Ser235, Met266, and Ala268 in the B transferase) (Seto et al., 1997Go, 1999; Yamamoto and McNeill, 1996Go) to determine if one, all, or a combination of these are important for determination of donor sugar specificity. To date there is no definitive answer because there is conflicting data from different groups as to exactly which residues are contributing. Yamamoto and McNeill (1996)Go suggest that residue 268 is required for conferring substrate specificity, whereas Seto et al. (1999)Go suggest residue 266. It is clear from these and other studies (Palcic et al., 2001Go), however, that amino acids 266 and 268 are the most important of the four for determining substrate specificity of the A and B transferases. The four amino acids in pig GT that correspond to the four changes in A and B transferase are Lys181, Trp240, His271, and Ala273.

Chemical modification can be used to identify amino acid side chains that are important for enzyme catalysis and function. Chemical modification of pig GT has suggested the importance of a His residue, as treatment with diethyl pyrocarbonate (DEPC) was sufficient to significantly decrease GT enzymatic activity and prevent the transfer of Gal from UDP-Gal to NAcLac (Stults et al., 1999Go). DEPC is known to modify His residues by the addition of an ethyl group to the imidazole ring (Lundblad, 1991Go), thereby rendering it inactive. The His271 residue of pig GT was considered a potential candidate for replacement because it corresponds to one of the four crucial amino acids in A and B transferases.

The crystal structure of bovine GT was recently solved (Boix et al., 2001Go; Gastinel et al., 2001Go) and was the first report of the structure of a mammalian retaining glycosyltransferase. The structure shows that 19 residues interact to form the catalytic pocket of GT, with 8 of these residues being invariant in the {alpha}-1,3Gal/GalNAc transferase family, suggesting their involvement in binding to the UDP moiety of the substrate sugar. Ten other residues were found to be conserved in all homologs of the GT family but are different in the A and B transferases as well as Forssman synthetase (Haslam and Baenziger, 1996Go; Keusch et al., 2000Go). This suggests that some of these residues may interact with the sugar moiety of the substrate molecule (UDP-Gal). These 10 residues correspond to Gln219, Gln238, Trp240, Trp241, Thr250, His271, Ala272, Ala273, Asp307, and Trp347 in the pig GT enzyme. Of these 10 residues, 3 correspond to the changes found in A and B transferase, Trp240, His271, and Ala273. The residues His271 and Ala273 are also found in iGb3 synthase, which is similar to GT in that it is capable of transferring Gal in an {alpha}-1,3 linkage (Keusch et al., 2000Go). These data, together with evidence discussed, strengthened the possibility of His271 being involved in catalysis and enzymatic function of pig GT, although neither of the two original crystal structures predicted the involvement of His271 (Boix et al., 2001Go; Gastinel et al., 2001Go).

Our approach was to replace His271 with an Ala residue, as well as with amino acids found in this position in other family 6 members that transfer GalNAc (A transferase and Forssman synthetase). Enzymes were then examined for their ability to transfer Gal in an {alpha}(1,3)-linkage to NAcLac and produce the Gal{alpha}(1,3)Gal epitope. Furthermore, we determined whether mutants containing amino acids at His271 found in GalNAc transferring enzymes, such as A transferase and Forssman synthetase, were able to transfer GalNAc to NAcLac, as other researchers have shown that changing this amino acid residue in A transferase can switch substrate specificity to that of B transferase. Thus, His271 was changed to a Leu (as in A transferase) and Gly (as in Forssman synthetase). Both transferases belong to the same glycosyltransferase family as pig GT and share high sequence identity (Haslam and Baenziger, 1996Go; Shetterly et al., 2001Go; Yamamoto and Hakomori, 1990Go). The A transferase and Forssman synthetase both transfer a terminal GalNAc moiety in an {alpha}-1,3 linkage from UDP-GalNAc to their respective acceptor molecules, H-substance (Hakomori, 1999Go) and globoside 4 (Gb4) (Haslam and Baenziger, 1996Go).

Here we report that pig GT enzymes containing a change at amino acid His271 to either an Ala, Leu, or a Gly residue were unable to transfer Gal to NAcLac. However, replacing His271 with another positively charged residue, Arg, retained the ability of the enzyme to transfer Gal to NAcLac and form the Gal{alpha}(1,3)Gal epitope. The interaction of His271 and the donor sugar substrate within the catalytic pocket was also analyzed by molecular modeling, which indicated that this reaction occurs via a molecular interaction between the positive charge of His271 and the Gal moiety of UDP-Gal. This functional data is supported at the molecular level by a recently accepted third crystallographic study of GT where the enzyme was cocrystallized with UDP-Gal (Boix et al., 2002Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Sequence analysis of the {alpha}-1,3Gal/GalNAc transferase family
Human A and B transferases provide an excellent model for examination of residues involved in donor substrate specificity of the {alpha}-1,3 Gal/GalNAc transferases. The change in donor substrate specificity of A and B transferase (GalNAc and Gal, respectively) can be attributed to four amino acid differences, which in pig GT correspond to Lys181, Trp240, His271, and Ala273 (Table I). The two residues shown to be critical in determining substrate specificity in A and B transferase (amino acids 266 and 268) (Seto et al., 1997Go, 1999; Yamamoto and McNeill, 1996Go) correspond to His271 and Ala273 in pGT. Ala273 corresponds to a Gly residue in A transferase, but this residue is also an Ala in B transferase, iGb3 synthase, and Forssman synthetase. His271 is conserved across all homologs of GT, as well as rat iGb3 synthase (which also creates an {alpha}(1,3)Gal linkage). In contrast, A transferase and Forssman synthetase have a Leu and Gly at position 271, respectively. These results suggested His271 was a potential candidate for determining donor substrate specificity of pig GT. To confirm and extend that His was important, we repeated the previously published chemical modification data using DEPC (Stults et al., 1999Go), HgCl2, and CuSO4, which are all capable of modifying His residues; all were able to significantly reduce GT activity (data not shown).


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Table I. Sequence alignment of the {alpha}(1,3)Gal/GalNAc transferase family
 
Replacing His271 with a neutral, nonpolar residue is sufficient to inhibit pGT activity
The initial pig GT mutant constructs replaced residue His271 with residues found in transferases that transfer an {alpha}-1,3GalNAc rather than an {alpha}-1,3Gal, to examine the effect of Gal transfer to NAcLac. pGTAA replaced His271 with an Ala residue, and pGTLA and pGTGA replaced His271 with a Leu and Gly, which resembled A transferase and Forssman synthetase, respectively (Table I). No constructs that resembled B transferase were made, because pig GT is very similar to the human B transferase in its ability to transfer a Gal sugar residue in an {alpha}-1,3 linkage to its acceptor molecule, H-substance.

Transfected COS cells were cell surface stained with Griffonia simplicifolia B4 (IB4)-fluorescein isothiocyanate (FITC) (Figure 1), which detects the Gal{alpha}(1,3)Gal epitope. As expected, cells expressing pGT stained positively for cell surface Gal{alpha}(1,3)Gal (Figure 1A), indicating the ability of the enzyme to transfer Gal to NAcLac. In constrast, transfection of constructs pGTAA, pGTLA, and pGTGA showed no expression of Gal{alpha}(1,3)Gal (Figure 1B, C, and D). These results were confirmed by enzymatic assay for {alpha}(1,3)GT using lysates from transfected cells. Pig GT transferred Gal at 102 nmol/mg/h, whereas all three mutant enzymes showed no significant activity above background (Table II). All three mutant enzymes have replaced residue 271 with a single, neutral, nonpolar amino acid; this result suggested that the presence of the His residue at this position is essential for pGT enzyme activity. To ensure that the effects seen were specific to His271, another His residue (His245) was also changed to Ala (pGTH245A) and exhibited normal GT activity (Figure 1E).



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Fig. 1. Mutation of His271 prevents cell surface expression of Gal{alpha}-1,3Gal. COS-7 cells were transfected with pGT and pGT mutant constructs, then stained for cell surface Gal{alpha}-1,3Gal with IB4-FITC. Cells were transfected with the following; A, pGT; B, pGTAA; C, pGTLA; D, pGTGA; E, pGTH245A; and F, pGTRA.

 

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Table II. Enzymatic activity of pGT and pGT mutant constructs
 
Replacing His271 with Arg restores GT activity
It was important to determine whether a substitution of residue His271 was affecting an interaction between the sugar and the amino acid side chain or whether it was simply involved in conformation of the catalytic pocket. His271 was therefore replaced with an Arg residue, another basic, charged amino acid. Arg differs from His in that it is more elongated and lacks the imidazole ring. Transfection of pGTRA produced Gal{alpha}(1,3)Gal on the cell surface, indicating that the ability to transfer Gal to the acceptor substrate NAcLac had been retained (Figure 1F) and that an interaction between Arg271 or His271 with the donor sugar substrate may be occurring.

Molecular modeling was used to examine the possible interaction of His271 with UDP-Gal, as well as the changes that occur in the catalytic pocket when His271 is replaced with either an Ala or Arg residue (Figure 2). In the wild type enzyme, the His271 side chain is shown to be sufficiently close and oriented in a manner suitable to interact with the Gal residue by attractive van der Waals forces and hydrogen bonding (Figure 2A). Replacement of His271 by Ala effectively eliminates these interactions, leaving a solvent-filled pocket adjacent to the Gal portion of the substrate (Figure 2C). When replacing His271 with an Arg residue, a low-energy side-chain rotamer was selected that resembled the conformation of the His residue in the bovine GT crystal structure (Figure 2B) (Gastinel et al., 2001Go). This conformation fitted snugly into the catalytic pocket with no unfavorable steric clashes with neighboring residues. Similar to His271, Arg was predicted to interact closely with the Gal residue by van der Waals interactions and hydrogen bonding through its guanidinium side chain (Figure 2B). Furthermore, complementarity for the substrate is obviously maintained with the Arg271 mutation, as is evident from the very similar molecular surfaces presented by the His and Arg side chains, as opposed to the Ala mutation (Figure 2C). These results support the data shown in the transfection studies, where both pGT and pGTRA were able to transfer Gal to the acceptor molecule NAcLac, whereas pGTAA was unable to catalyze the reaction (Figure 1).



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Fig. 2. Molecular modeling of the catalytic pocket of pGT, pGTAA, and pGTRA. Models of amino acid residues involved in forming the catalytic pocket of A, pGT; B, pGTRA; and C, pGTAA. UDP-Gal is shown in blue-green.

 
It was necessary to ensure that lack of GT activity in constructs pGTAA, pGTLA, and pGTGA was not due to impaired translation, expression, or folding of the enzymes. FLAG-tagged enzymes were expressed in COS-7 cells and intracellular localization was detected with an anti-FLAG monoclonal antibody (Figure 3). All mutant enzymes, including those that no longer showed GT activity, could be detected within the cells with the anti-FLAG antibody and were shown to have normal Golgi-like localization.



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Fig. 3. Intracellular expression of pGT and pGT mutant constructs. COS-7 cells were transfected with pGT and pGT mutant constructs, permeabilized, and stained intracellularly for the FLAG epitope. Cells were transfected with the following: A, pGT; B, pGTAA; C, pGTLA; D, pGTGA; E, pGTRA; and F, pGTH245A. Positively stained cells showed the perinuclear staining pattern indicative of Golgi localization, as would be expected of pGT expression.

 
Replacing His271 with residues from {alpha}-GalNAc-transferring enzymes does not result in transfer of GalNAc to NAcLac
Having shown that production of Gal{alpha}(1,3)Gal could be inhibited by replacing His271 with a residue from either A transferase or Forssman synthetase, the possibility of change in substrate specificity of the enzymes from Gal to GalNAc was examined. Transfected cells were stained with lectins that detect different carbohydrate epitopes (and therefore different enzyme activities) that have been previously shown to be far more sensitive than radioactive enzyme assays (Yamamoto and McNeill, 1996Go). IB4 lectin is capable of detecting the Gal{alpha}(1,3)Gal carbohydrate epitope (Wu et al., 1995Go), as seen in the previous data (Figure 1). Griffonia simplicifolia A4 (IA4) lectin shows a preference for terminal {alpha}(1,3)GalNAc epitopes but is also capable of detecting {alpha}(1,3)Gal (Wu et al., 1999Go). Helix pomatia (HPA) lectin is specific for {alpha}(1,3)GalNAc (Matsui et al., 2001Go) and was used to refine whether results obtained with IA4 were indeed {alpha}(1,3)GalNAc rather than {alpha}(1,3)Gal. Because COS-7 cells constitutively express {alpha}(1,3)GalNAc when stained with both IA4 and HPA lectins (data not shown; Clarke and Watkins, 1999Go), they were not suitable for determining if pGT mutants were able to transfer {alpha}(1,3)GalNAc to NacLac and express this epitope (GalNAc{alpha}(1,3)Galß(1,4)GlcNAc) on the cell surface. Therefore Chinese hamster ovary cells transformed with polyoma large T antigen (CHOP cells), which are {alpha}(1,3)GalNAc negative, were used to determine if the mutant constructs were expressing a terminal {alpha}(1,3)GalNAc epitope on the cell surface (Figure 4).



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Fig. 4. Determination of pGT and pGT mutants substrate specificity to {alpha}1,3Gal and {alpha}1,3GalNAc. CHOP cells were transfected with the following DNA constructs: pGT, pGTAA, pGTLA, pGTGA, pGTRA, and pGTH245A. The cells were analyzed by flow cytometry after surface staining with IB4-FITC (AF) , IA4-FITC (GL), and HPA-FITC (MR). Unstained cells are shown as a thin line, and stained cells are represented by a bold line.

 
Pig GT and constructs containing single mutations (pGTAA, pGTLA, PGTGA, and pGTRA) were transfected into CHOP cells. Cell surface expression of Gal{alpha}(1,3)Gal was detected by IB4 (Figure 4A–E) or IA4 (Figure 4F–J). Expression of {alpha}(1,3)GalNAc was detected by HPA (Figure 4K–O). In this transient expression system, 100% transfection efficiency is unachievable, thus a negative peak of 5 mean fluorescence units (mfu) for IB4 and HPA and 10 mfu for IA4 is usually observed (Figure 4), with the positive peak falling above these values. As expected, transfection of pGT produced the Gal{alpha}(1,3)Gal epitope on the cell surface as detected by IB4 (Figure 4A), where positive cells had 259 mfu. However, cells transfected with constructs pGTAA, pGTLA, and pGTGA did not stain with IB4 (Figure 4B, C, and D), indicating that Gal{alpha}(1,3)Gal was not produced and that {alpha}(1,3)GalNAc was not transferred to NAcLac. Cells transfected with pGTRA stained positively for IB4 (Figure 4E), indicating cell surface expression of Gal{alpha}(1,3)Gal; however the level of fluorescence was decreased (55 mfu) compared to wild-type pig GT. Although this result may suggest that an Arg at position 271 is not as efficient as the wild-type enzyme, it is still capable of transferring a Gal residue to NAcLac, therefore retaining its substrate specificity for the Gal donor sugar. However, further experiments may be required to characterize the substrate affinity of the mutant enzymes compared to wild-type GT. The results confirm the microscopy data (Figure 1), where IB4-FITC was used to visualize the cell surface expression of Gal{alpha}(1,3)Gal.

The IA4 lectin shows highest specificity for {alpha}(1,3)GalNAc, but it can also detect {alpha}(1,3)Gal with lower affinity. Therefore, cells expressing pGT would be expected to bind both IB4 and IA4 lectins. Mutant constructs, however, would not bind IB4 but would be able to bind IA4 if the donor sugar specificity had been altered to allow the transfer of GalNAc to NAcLac. Transfection with pGT produced Gal{alpha}(1,3)Gal on the cell surface, which was detected by IA4, where positive cells had 171 mfu (Figure 4F). Cells transfected with pGTAA, pGTLA, and pGTGA did not stain with IA4 (Figure 4G, H, and I) indicating that there had been no transfer of a terminal {alpha}(1,3)GalNAc to NAcLac. Construct pGTRA behaved like pGT, with cells staining positively with IA4 (Figure 4J), however, the level of fluorescence observed was weak.

HPA lectin was used to confirm results obtained with IA4. Cells transfected with pGT did not react with HPA (Figure 4K), because no {alpha}(1,3)GalNAc was present on the cell surface. Constructs pGTAA, pGTLA, and pGTGA did not stain with HPA (Figure 4L, M, and N), thus confirming that the mutant constructs were unable to transfer GalNAc to the acceptor molecule NAcLac. Transfection of construct pGTRA did not produce a carbohydrate epitope containing terminal {alpha}(1,3)GalNAc and was therefore negative for HPA staining (Figure 4O).

Although the single amino acid substitutions at position 271 described did not switch donor substrate specificity from Gal to GalNAc, position 273 was also substituted with amino acids present in GalNAc-transferring enzymes because two substitutions may be more likely to switch the specificity. Constructs pGTLG and pGTGG, which contained the double substitutions at amino acid positions 271 and 273 (Gly as occurs in A transferase), were also transfected into CHOP cells and analyzed for cell surface expression of {alpha}(1,3)Gal and {alpha}(1,3)GalNAc. The results observed were similar to constructs with a single change at position 271. Cells transfected with the constructs did not stain with IB4, IA4, or HPA (data not shown), indicating a lack of production of Gal{alpha}(1,3)Gal and no transfer of GalNAc to NAcLac. Construct pGTRG behaved like pGT, indicating that residue 273 by itself was insufficient to alter enzyme activity or prevent the production of Gal{alpha}(1,3)Gal (data not shown). These results confirm the COS-7 cell data, showing that pig GT produces the Gal{alpha}(1,3)Gal carbohydrate epitope and that substituting His271 with Arg (pGTRA) can partially retain the ability of the enzyme to transfer Gal to NAcLac. Constructs pGTAA, pGTLA, and pGTGA were unable to produce the Gal{alpha}(1,3)Gal epitope; however, we have also shown that they did not become more like A transferase and were unable to transfer either an {alpha}-1,3Gal or an {alpha}-1,3GalNAc to the acceptor substrate, NAcLac.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It is estimated that over 200 glycosyltransferases are required to generate all known glycosidic linkages (Opat et al., 2001Go). It has yet to be determined how glycosyltransferases create these glycosidic linkages or how donor and acceptor specificity is determined. Here we show that His271 of pig GT is crucial for enzymatic activity to create the Gal{alpha}(1,3)Gal carbohydrate epitope by transferring Gal to NAcLac. Furthermore, constructs containing an Arg at position 271 (pGTRA and pGTRG) were also able to utilize UDP-Gal (Figures 1 and 4). Molecular modeling was used to show an interaction of amino acid 271 with the Gal moiety of UDP-Gal, where both His and Arg were sufficiently close and oriented in such a way that a direct interaction can occur. In contrast, an Ala at this position eliminates the possibility of an interaction. The importance of His271 was not predicted by the first two crystal structures of the bovine GT enzyme (Boix et al., 2001Go; Gastinel et al., 2001Go). We have also shown that substituting residues 271 and 273 of pGT to residues that are present in GalNAc utilizing enzymes A transferase and Forssman synthetase was not sufficient to allow transfer of GalNAc to the acceptor, NAcLac.

The lack of enzymatic function of the GT mutants is either due to (1) lack of proper folding of the protein or (2) involvement of the altered residue in either substrate/donor carbohydrate recognition or indeed catalysis. The first possibility can only be properly determined by solving the 3D structure of the mutant proteins; however, misfolding of the proteins can be excluded by several criteria. First the mutant transferases correctly localize to the Golgi (Figure 3). If the proteins were misfolded it is likely that they would be retained in the endoplasmic reticulum prior to transport to the proteosome, as has been shown for misfolded proteins (Graves et al., 2001Go; Halaban et al., 2001Go; Helenius and Aebi, 2001Go; Toyofuku et al., 1999Go). Second, the amino acids chosen as replacements are those found in other functional family members (Table I), thus they have been selected during evolution to be suitable at this position. Third, single amino acid substitutions have been extensively used to characterize residues required for enzymatic activity of glycosyltransferases (Ihara et al., 2002Go; Laroy et al., 2001Go; Li et al., 2001Go; Malissard et al., 2002Go) and other proteins (Handschuh et al., 2001Go; Zaganas and Plaitakis, 2002Go). Finally, molecular modeling did not show any major changes in structure after the amino acid replacements.

The crystal structure of bovine {alpha}-1,3GT has provided a great deal of information regarding the conformation of the enzyme. However, functional studies are also required to determine the exact residues which interact with the Gal moiety of UDP-Gal and the acceptor substrate. Of the 19 residues that make up the catalytic pocket for donor sugar binding, 8 are found to be invariant across the {alpha}(1,3)Gal/GalNAc transferase family, and these are thought to bind to UDP (Gastinel et al., 2001Go). It has also been proposed that one of these amino acids may provide the residue responsible for nucleophilic attack on the C1 atom of the transferred sugar (Gastinel et al., 2001Go). Ten other residues in the catalytic pocket are conserved among all homologs of GT, and therefore one or more of these residues may be able to confer donor sugar specificity. Residues His271 and Ala273 (referred to as His280 and Ala282 in bovine GT) fall into this group of amino acids, and the crystal structure shows that they are located in the middle of the ß-sheet, which forms the bottom of the catalytic pocket (Gastinel et al., 2001Go). It was suggested that His271 may be involved in retaining the size and conformation of the catalytic pocket rather than directly interacting with the Gal moiety of UDP-Gal (Gastinel et al., 2001Go), and the side chain of Ala273 is thought to be pointing upward from the pocket and mutation to a residue, such as Gly (as in A transferase; Figure 1D), would create steric hindrance and prevent entry of UDP-Gal into the pocket (Gastinel et al., 2001Go).

We have shown with functional data that replacing His271 alone is sufficient to prevent enzymatic activity, which suggests that this residue is crucial for catalysis. Molecular modeling was then used to confirm these results and show that His271 was capable of interacting with UDP-Gal. The models illustrated in Figure 2 show a direct interaction between His271 and the Gal residue (Figure 2A), which is probably maintained with the Arg side chain (Figure 2B) but not when the side chain is reduced to a beta-carbon atom as with the Ala mutation (Figure 2C). The models indicate that replacement of His271 by Ala has no significant effect on the spatial arrangement of neighboring residues in the catalytic pocket. Thus the inactivity of the pGTAA mutant probably results from a loss of steric compatibility and/or positive charge of the subsite, which is occupied by the Gal residue. In contrast, Arg271 (Figure 2B) was predicted to occupy a similar volume as the His residue (Figure 2A) as well as presenting a positively charged planar guanidinium group to the Gal residue. Replacing His with Arg also did not alter the conformation of the remaining residues within the catalytic pocket. Considered together, the models suggest that both the steric bulk and the positive potential on the side chain of His271 are important for maintaining the catalytic ability of pig GT. Thus His271 may have a more central role in UDP-Gal catalysis than simply maintaining the shape of the catalytic pocket, as suggested previously (Boix et al., 2001Go; Gastinel et al., 2001Go). Direct structural evidence supporting the critical role of His271 comes from the work of Boix et al. (2002)Go, a report accepted for publication during the revision of this manuscript. These authors report the interaction of His280 of bovine GT (corresponding to His271 of pig GT) via a hydrogen bond between the side chain of His and O2 of the Gal moiety of UDP-Gal.

Another possibility for the involvement of His271 in catalysis of pig GT is an interaction with a Mn2+ ion. His residues are often involved in coordinating the binding of divalent cations; however, this possibility is unlikely for His271. First, residues in the A and B transferases that correspond to His271 of pGT have been shown to be involved in the binding of donor sugar substrate, rather than binding of the divalent cation (Seto et al., 1999Go; Yamamoto and McNeill, 1996Go). Furthermore, although it has been suggested that at least two activating metal-binding sites are present in this enzyme (Shah et al., 2000Go; Zhang et al., 2001Go), it has been shown that most retaining glycosyltransferases coordinate binding of the divalent cation through the DXD motif, which is found toward the N-terminal region of the catalytic domain (Li et al., 2001Go; Rao and Tvaroska, 2001Go). We have also shown that replacing His271 with an Arg residue is sufficient to retain activity of pig GT, and Arg is not known to be a divalent cation coordinating residue due to its strong positive charge. Last, molecular modeling of the catalytic pocket and energy minimalization was based on the crystal structure of bovine GT, where the corresponding residue, His280, was not shown to be involved in any metal-binding events (Boix et al., 2001Go; Gastinel et al., 2001Go). Therefore, it seems likely that a direct interaction is occurring between the sugar and the positive charge of either His or Arg, rather than a conformational role where His would be retaining the shape of the pocket (Gastinel et al., 2001Go). This proposal is further supported by the replacement of His271 with Leu. Leu has a very similar side chain to Arg, missing only the charged amino group at the end of the residue, and would therefore fill a similar space within the catalytic pocket. However, the His271 Leu mutant (pGTLA) was unable to produce Gal{alpha}(1,3)Gal, showing that a direct interaction of the amino acid and a positive charge is required.

The second amino acid of the A and B transferases, which has been implicated in substrate specificity, is residue 273 (268 in A and B transferase). Constructs containing a double mutation at positions 271 and 273 were analyzed for their ability to produce Gal{alpha}(1,3)Gal as well as their ability to transfer an {alpha}(1,3)GalNAc residue to NAcLac. The pGTRG construct was capable of creating Gal{alpha}(1,3)Gal, suggesting that Ala273 does not play a critical role in enzymatic activity and that replacing Ala with a Gly residue does not block the entry of UDP-Gal into the catalytic pocket by steric hindrance. However, it is not surprising that Ala273 alone does not induce a change in substrate binding, because this residue is conserved across all members of the {alpha}-(1,3)Gal/GalNAc transferase family, with the exception of A transferase (Table I).

It was expected that changing His271 alone or in combination with Ala273 to residues found in the A transferase or Forssman synthetase may switch the donor substrate specificity from UDP-Gal to UDP-GalNAc. However, carbohydrate residues containing a terminal {alpha}(1,3)GalNAc were not observed after transfection of mutants into CHOP cells. In addition, {alpha}(1,3)GalNAc activity was not detected by enzyme assay of transfected CHOP cell lysates (data not shown). It has recently been shown that the order of donor sugar and acceptor substrate binding occurs sequentially and that the donor sugar substrate binding occurs before the acceptor substrate (Zhang et al., 2001Go). The binding of the donor sugar substrate can alter the conformation of the enzyme and then create an acceptor binding domain (Zhang et al., 2001Go). Thus, one possibility is that mutant pig GT may bind UDP-GalNAc instead of UDP-Gal and change the conformation of the acceptor binding domain such that NAcLac can no longer bind, thereby rendering the enzyme inactive.

It still remains for researchers to examine how acceptor substrate binding is affected by binding of the donor sugar and which part of the transferase molecule is interacting with the acceptor substrate. In addition, further studies are required to determine whether the mutant pig GT constructs are capable of binding another sugar substrate such as GalNAc, and if so, why the transfer to the acceptor molecule is inhibited.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
DNA constructs
Eleven pGT mutants were generated using splice overlap extension–polymerase chain reaction (PCR) (Horton et al., 1989Go) using pGT DNA (Dabkowski et al., 1993Go) as template. The PCR primers used and nucleotide positions with which they anneal are shown in Table III. Each construct contained either one or two amino acid substitutions corresponding to wild-type amino acid positions His271 and/or Ala273 (except for pGTH245A, which contained a substitution of His with Ala at residue 245). For example, construct pGTAA contains an Ala271 and Ala273, whereas construct pGTLG contains a Leu271 and Gly273 (Table IV). The final fragments generated contained the appropriate point mutations with BamHI and XbaI sites at their 5' and 3' ends, respectively. All restriction and modifying enzymes were from Biolabs, and DNA was purified on Qiagen Gel Extraction Columns (Qiagen, Holden, Germany). For localization studies, pGT, pGTAA, pGTLA, pGTGA, pGTRA, and pGTH245A constructs were tagged with the FLAG epitope (DYKDDDDKR) at the carboxy terminus. PCR reactions were carried out in a Hybrid DNA Thermal Cycler using Pwo DNA Polymerase (Roche, Basel, Switzerland). BamHI and XbaI digested PCR fragments were ligated into pcDNA1 (Invitrogen, Groningen, the Netherlands). Plasmid DNA was prepared using the Maxiprep DNA Purification System (Qiagen). Cloning site and sequence integrity was confirmed by DNA sequencing.


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Table III. Oligonucleotide primers used to create pGT mutant constructs
 

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Table IV. pGT mutant constructs created by splice overlap extension PCR
 
Transfection, serology, and enzyme assays
COS-7 and CHOP cells (Heffernan and Dennis, 1991Go) were cultured in Dulbecco’s modified Eagle’s medium (CSL, Melbourne, Australia) supplemented with 10% fetal calf serum. Cells were transfected using LipofectAMINE Plus reagent (Life Technologies, Rockville, MD) as recommended by the manufacturer, then examined after 48 h for cell surface expression of carbohydrate epitopes using lectins as a measure of enzyme activity. Lectins are an extremely sensitive method for detection of enzyme activity (Yamamoto and McNeill, 1996Go) and also provide more reproducible results than radioactive enzyme assays. Gal{alpha}(1,3)Gal was detected with FITC-labeled IB4 and IA4 lectins. Terminal {alpha}(1,3)GalNAc were detected with IA4 and HPA lectins (Sigma, St. Louis, MO). Fluorescence was detected by microscopy and flow cytometry (Becton Dickinson FACScalibur, Franklin Lakes, NJ).

For intracellular localization of the expressed glycosyltransferases, DNA encoding the FLAG-tagged enzymes were transfected into COS-7 cells. Expression of tagged protein was determined after 48 h. Cells were fixed in phosphate buffered saline (PBS)/2% paraformaldehyde for 10 min and then permeabiliszd in PBS/0.5% saponin for 10 min, followed by staining with either IB4-FITC or anti-FLAG BioM2 antibody (Sigma) followed by streptavidin-alexa594 (Molecular Probes, Eugene, OR). Intracellular staining was detected using a confocal laser scanning microscope (Optiscan, Melbourne, Australia).

CHOP cells were transfected with constructs and lysates were assayed for enzyme activity as previously described (Sandrin et al., 1995Go).

Homology modeling
The 2.3 Å crystal structure of bovine {alpha}-1,3GT (Gastinel et al., 2001Go; PDB no. 1G93) was used as a template to construct a homology model of pGT. A sequence alignment based on residue identity and similarity, with any gaps/insertions placed outside structurally conserved regions, was prepared for the bovine and porcine GT enzymes. Using this alignment, an atomic model of pGT was constructed from constraints extracted from the template coordinates. For identical or similar residues, side chains were positioned starting with the conformations observed in the {alpha}-1,3GT crystal structure. The remaining side-chain conformations were generated automatically from a rotamer library, with rotamers selected that minimized steric clashes with neighboring residues. Residue His271 was substituted with Ala or Arg to prepare the pGTAA and pGTRA models. Models were optimised by molecular dynamics and energy-minimization with the GROMOS96 43B1 implementation of Swiss-pdb viewer (Guex and Peitsch, 1997Go). Figures were prepared with the program MOLMOL (Koradi et al., 1996Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We would like to thank Bruce Wines, Bruce Stone, Dale Christiansen, and Bach-Tuyet Lam for advice and helpful comments. This work was supported by the National Health and Medical Research Council of Australia.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CHOP, Chinese hamster ovary cells transformed with polyoma large T antigen; DEPC, diethyl pyrocarbonate; FITC, fluorescein isothiocyanate; GT, {alpha}(1,3)galactosyltransferase; HPA, Helix pomatia; IA4, Griffonia (Bandeiraea) simplificola lectin 1A; IB4, Griffonia (Bandeiraea) simplificola lectin 1B; PBS, phosphate buffered saline; PCR, polymerase chain reaction.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: m.sandrin@ari.unimelb.edu.au Back


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 Introduction
 Results
 Discussion
 Materials and methods
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 References
 
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