Xenotransplantation is now considered as the main potential remedy for the shortage of donor organs (see Nature, special issue 391, 1998). However, when a pig organ is transplanted in humans, a hyperacute vascular rejection of the graft occurs because human preformed antibodies recognize [alpha]Gal(1-3)[beta]Gal terminal carbohydrates present on porcine endothelial cells (Galili, 1991; Cooper et al., 1994). This so-called xeno-antigen is present on the cells of most mammals with the exception of humans and Old World monkeys. The UDP-Gal:[beta]1-4GlcNAc-R [alpha]3-galactosyltransferase ([alpha]3-GalT, EC 2.4.1.151) is the enzyme responsible for the formation of the [alpha]-Gal epitope and is therefore the subject of high interest in the field of xenotransplantation. Having access to the 3D structure of this enzyme would open new routes for the design of inhibitors, that could act in vitro or in vivo. It will eventually also allow for engineering the enzyme in the aim of producing xeno-oligosaccharides or related ones. Recently, recombinant bovine [alpha]3-GalT has been used for chemoenzymatic synthesis of the [alpha]-Gal epitope (Joziasse et al., 1990; Fang et al., 1998). These oligosaccharides can be immobilized on an affinity column for depleting the anti-Gal antibodies of the recipient (Taniguchi et al., 1996).
Several mammalian [alpha]3-GalT genes have been cloned, i.e., from pig (Strahan et al., 1995), cow (Joziasse et al., 1989), mouse (Joziasse et al., 1992), and marmoset (Henion et al., 1994). As all the other Golgi-resident eukaryotic glycosyltransferases, these enzymes are type-II membrane proteins consisting in a short N-terminal cytosolic tail, a transmembrane region, a stem, and a C-terminal catalytic domain. Amino acid sequence similarities have been found between the [alpha]3-GalTs and others [alpha]3-GalNAc- and [alpha]3-Galtransferases that use different oligosaccharide acceptors (Yamamoto et al., 1990; see Table I). Blood group A and B transferases, which makes [alpha]3-GalNAc- and [alpha]3-Gal linkages, respectively, require fucosylated N-acetyllactosamine as acceptor, whereas the Forssman synthase (Haslam and Baenziger, 1996) requires [beta]-GalNAc. More distant amino acid sequences homologies have been evidenced with [beta]4-GalTs (Joziasse et al., 1989; Breton et al., 1998).
In the absence of crystal structure of glycosyltransferases, the aim of the present work is to extract structural information from the amino acid sequences. Since [alpha]3-GalTs are not homologous to any proteins of known 3D-structures, the first step is to search for proteins that could share the same fold. Fold recognition is a theoretical approach which allows the alignment of one sequence with one structure by a process referred to as "threading" (Lemer et al., 1995). In practice, a library of known 3D structures is searched to determine the folds that gives the best alignments with the sequence of interest. Threading the sequence against all possible folds, and then sorting and ranking the possible solutions form the three steps of a fold recognition study. Several programs are available to carry out such a process and can be classified into two families based on their algorithms: the prediction-based methods align the predicted secondary structure of the searched sequence with the secondary structure elements from known crystal structures (Rost et al., 1997), whereas potential-based methods use mean force potentials derived from a database of known structures (Jones and Thornton, 1996; Vajda et al., 1997; Rooman and Gilis, 1998). Recently, such approaches were successfully used to predict the fold of the C-terminal lectin-like domain of polypeptide-GalNAc transferases (Imberty et al., 1997). Here we have applied these methods to the pig [alpha]3-GalT sequence, the enzyme responsible for the biosynthesis of the xeno-antigen, and have used homology modeling to build the nucleotide-binding domain of this protein.Table I.
Abbreviation | Name | Origin | aab | Product | Accession number |
Pig [alpha]-GalT | [alpha]-Galactosyltransferase | Sus scrofa | 371 | [alpha]Gal(1-3)[beta]Gal(1-4)[beta]GlcNAc-R | L36152 |
L36535 (clone pPGT-3) | |||||
Bov [alpha]-GalT | [alpha]-Galactosyltransferase | Bos taurus | 368 | [alpha]Gal(1-3)[beta]Gal(1-4)[beta]GlcNAc-R | J04989 |
Mar [alpha]-GalT | [alpha]-Galactosyltransferase | Callithrix sp. | 376 | [alpha]Gal(1-3)[beta]Gal(1-4)[beta]GlcNAc-R | S71333 |
Mou [alpha]-GalT | [alpha]-Galactosyltransferase | Mus musculus | 368 | [alpha]Gal(1-3)[beta]Gal(1-4)[beta]GlcNAc-R | M85153 |
M26925 | |||||
Pig bgA | [Agr] Transferase | Sus scrofa | 364 | [alpha]GalNAc(1-3)[[alpha]Fuc(1-2][beta]Gal-R | AF050177 (hypothetical) |
Dog Forss | Forssman synthase | Canis familiaris | 347 | [alpha]GalNAc(1-3)[beta]GalNAc-R | U66140 |
Hum bgAa | A Transferase | Homo sapiens | 354 | [alpha]GalNAc(1-3)[[alpha]Fuc(1-2][beta]Gal-R | J05175 (A34933, P16442) |
X84746 (complete cDNA) | |||||
338 | Y11891 (synthetic) | ||||
Hum bgBa | B transferase | Homo sapiens | 354 | [alpha]Gal(1-3)[[alpha]Fuc(1-2][beta]Gal-R | 1609195 (PRF databank) |
338 | X91874 (synthetic) |
Table II.
Bov [alpha]-GalT | Mar [alpha]-GalT | Mou [alpha]-GalT | Hum bgB | Hum bgA | Pig bgA | Dog Forss | |
Pig [alpha]-GalT | 84.1 | 82.4 | 74.8 | 36.9 | 36.8 | 36.1 | 36.3 |
88.6 | 86.4 | 79.9 | 43.8 | 43.4 | 43.4 | 42.1 | |
Bov [alpha]-GalT | 82.4 | 73.6 | 37.4 | 37.8 | 36.4 | 36.6 | |
88.3 | 80.2 | 45.3 | 44.9 | 44.2 | 43.6 | ||
Mar [alpha]-GalT | 74.8 | 36.4 | 35.8 | 36.4 | 33.8 | ||
79.9 | 45.3 | 45.3 | 43.8 | 41.4 | |||
Mou [alpha]-GalT | 39.9 | 39.6 | 38.5 | 36.9 | |||
48.9 | 48.9 | 46.4 | 44.0 | ||||
Hum bgB | 96.9 | 62.8 | 42.8 | ||||
97.8 | 74.4 | 50.2 | |||||
Hum bgA | 63.0 | 43.3 | |||||
75.4 | 50.0 | ||||||
Pig bgA | 44.1 | ||||||
51.5 |
Sequence alignment and secondary structure prediction
Pairwise sequence alignment was performed for all [alpha]3-GalTs and blood group transferases sequences and the resulting scores are listed in Table II. Both complete and truncated sequences were considered since the transmembrane and stem regions display higher sequence variation than the catalytic domain. All the [alpha]3-GalT sequences are very similar, with identity scores ranging from 80 to 89% when the variable stem is omitted from the analysis. Blood group A and B transferases form another homogenous group, the pig bgA sequence being 75% identical with the human enzymes. The Forssman synthase is closer to the A and B transferases (about 50% identity) than to the [alpha]3-GalT (about 42% identity). Nevertheless, when looking at the catalytic domain, the identity score between all these enzymes is always better than 40%, and therefore they could be aligned with little difficulty.
Figure
Figure 1. Multiple alignment of [alpha]3-GalT and blood group transferase sequences. The numbering above refers to the pig [alpha]3-GalT sequence. The different shades of background with white text differentiate the hydrophobic, basic, acidic, and small side-chains amino acids. Pro and Gly have a black background, whereas Trp and Cys are indicated with black text on a white and gray background, respectively. Consensus sequence is given under the alignment: boldface capitals letters are used for fully conserved amino acids and numbers for conserved properties (1 for acidic, 2 for small side chain, 3 for basics, 4 for aromatic, and 5 for hydrophobic). Lowercase letters are used for amino acids with partial conservation. The secondary structure predictions from several programs are also displayed with H for [alpha]-helix and E for [beta]-strand. When a consensus is obtained, the letter is indicated in boldface.
The secondary structure predictions obtained using several programs are also displayed in Figure Fold recognition studies of [alpha]3-galactosyltransferases.
We present here the results obtained for [alpha]3-GalTs using the ProFIT program that is based on an energy-function potential (Flöckner et al., 1995, 1997; Sippl and Flöckner, 1996). Two databases of 3D-structures were created for the threading process, in addition to the default one provided with the program. The first one contains 105 3D-structures corresponding to almost all carbohydrate-interacting enzymes (glycosylhydrolases, lectins, toxins, CGTases . . .) whereas the second contains 38 structures representative of nucleotide-binding proteins. All the mammalian [alpha]3-GalTs sequences were used for the threading calculations. The transmembrane and stem regions were not considered. For each sequence, the best hits from the carbohydrate- and the nucleotide-binding proteins databanks are listed in Table III and Table IV, respectively. The class of fold and the topology are described as given in the SCOP database (Murzin et al., 1995). All but two of the predicted folds are composed of a mixture of [alpha]-helices and [beta]-strands. Most of them belong to the ([alpha]/[beta]) class, therefore consisting of alternating [alpha] and [beta] elements, a result which is in agreement with the secondary structure prediction (Figure
Table III.
Table IV.
When taking into account the number of hits and values of score, the final ranking of the prediction is: (1) one or two domains consisting of a [beta]-sheet with parallel strands surrounded on each side by [alpha]-helices connecting each strand (described as three layers [alpha]/[beta]/[alpha] in Table III and IV), (2) the classical ([alpha]/[beta])8 TIM-barrel consisting in a parallel [beta]-sheet closed barrel surrounded by connecting helices, and (3) other types of [alpha]/[beta]/[alpha] layers but with mixed orientations of the [beta]-strands. In the final step, when looking for the best candidate, one should also take into account the binding and catalytic function of each candidate protein. All of the nucleotide phosphorylases, synthases, and elongation factors listed in Table IV have the ability to bind nucleotide diphosphate but do not have any specificity for carbohydrate. In Table III, all proteins can bind carbohydrate but only the [beta]-glucosyltransferase from phage T4 ([beta]-GlcT) also contains a nucleotide-binding domain. This enzyme consists of two non-identical domains that adopt similar topology: a [beta]-sheet of five or six parallel strands surrounded by [alpha]-helices (Vrielink et al., 1994). The [beta]-GlcT catalyzes the transfer of glucose from UDP-glucose to hydroxymethylcytosine in modified DNA (Kornberg et al., 1961). Therefore, based on both the fold recognition study and similarity of function, [beta]-glucosyltransferase from phage T4 probably represents the best candidate for the fold of [alpha]3-GalTs. Molecular modeling of the C-terminal domain
Since the common feature between the phage [beta]-GlcT and the mammalian [alpha]3-GalTs is the use of a UDP-sugar, it can be hypothesized that their 3D similarities will be stronger in the nucleotide-sugar binding domain. In the crystal structure of [beta]-GlcT/UDP complex, the nucleotide sugar binding domain (or at least the nucleotide binding domain) has been identified to be the classical Rossman fold corresponding to the C-terminal domain (Vrielink et al., 1994). The catalytic amino acids cannot be identified, since the mechanism is still unknown. As a consequence of the fold recognition study above, it is predicted that the C-terminal region (about 150 amino acids) of mammalian [alpha]3-GalTs will act as nucleotide-sugar domain, and this is therefore only this domain that can be built by homology methods.
Homology modeling methods can be applied in such a case. Several steps of which are detailed below. In the present case, the main problem was producing a satisfactory alignment between the targeted pig [alpha]3-GalT sequence and a sequence of known 3D-structure. The advantages and limits of threading methods are now well identified. They are able to predict the correct protein fold from a sequence with reasonable confidence but encounter difficulties in producing correct sequence alignments between the sequence of interest and the known structure (Jones and Thornton, 1996). Since no homology for [alpha]3-GalT could be detected by classical alignment methods, a method based on the comparison of hydrophobic structural motifs was used, namely the HCA method (Gaboriaud et al., 1987). Structural homologues for the [beta]-glucosyltransferase
When the crystal structure of [beta]-GlcT was first published, it was assessed that this special topology consisting of the combination of two Rossman folds connected by an extended loop is not shared by other protein structures (Vrielink et al., 1994). Subsequently, several powerful algorithms for identifying similarities in 3D structures were developed and the entire structure of [beta]-GlcT was demonstrated to be topographically equivalent to the catalytic core of the much larger glycogen phosphorylase (GP) (Artymiuk et al., 1995; Holm and Sander, 1995). More interestingly, when looking at the crystal structure of glycogen phosphorylase complexed with pyridoxal 5[prime]-phosphate (PLP) coenzyme (Acharya et al., 1991), there is a striking similarity in the spatial arrangement of this substrate when compared with UDP in [beta]-GlcT. In most of the classifications now available for protein topologies, [beta]-GlcT from phage T4 is clustered with glycogen phosphorylase from rabbit and yeast and with the maltodextrin phosphorylase from E.coli. In the present study, we used the 3D alignment between [beta]-GlcT (2BGU) and one of the 22 rabbit glycogen phosphorylase (1GPB) proposed in the CAMPASS database (Sowdhamini et al., 1998). Despite a very low sequence identity score (10.6%), all of the secondary structure elements of the [beta]-GlcT can be aligned with those in the GP structure. Model of the C-terminal domain of pig [alpha]-3GalT
Knowledge-based modeling methods are based on the definition of structurally-conserved regions (SCR) in a family of similar structures, that can be reproduced in the target sequence. In the structural superfamily of [beta]-GlcT/GP, four crystal structures were selected to serve as the basis of the homology modeling procedure: rabbit glycogen phosphorylase (1GPB) (Acharya et al., 1991), phosphorylated form of yeast glycogen phosphorylase (1YGP) (Lin et al., 1996), E.coli maltodextrin phosphorylase (1AHP) (O'Reilly et al., 1997), and [beta]-glucosyltransferase from phage T4, that corresponds to the published crystal structure (2BGT) (Vrielink et al., 1994) but with determination of the loops that were missing in the first crystal structure (Morera and Freemont, personal communication). The three glycogen phosphorylases selected here are complexed with pyridoxal-5[prime]-phosphate.
The HCA method was used to generate a correct alignment between the pig [alpha]3-GalT and the nucleotide-binding domain of the structures from the library (Figure
Figure 2. HCA alignment of the C-terminal domain of [alpha]3-GalT, [beta]-GlcT, and GP. The one-letter code is used for amino acids except for Gly, Pro, Thr, and Ser, which are represented by a diamond, a star, a square, and a square with a dot, respectively. Conserved hydrophobic clusters have been shaded. The limits of the [alpha]-helices and [beta]-strands of the [beta]-GlcT and GP structures are indicated by vertical lines. In these enzymes, the amino acids directly involved in phosphate recognition are given as white text on gray backgrounds, whereas the ones involved in binding other part of the substrate have a black background. The deduced topology of [alpha]3-GalT is indicated and the secondary elements are numbered following the [beta]-GlcT structure.
A 3D model of the pig [alpha]3-GalT C-terminal domain was built using the COMPOSER program (Blundell et al., 1988). Nine structurally conserved regions (SCRs) were defined, each of them including one or two of the secondary structure elements. After local geometry optimization of the loops, the model does not display any stereochemical defects. According to the PROCHECK program (Laskowski et al., 1993), no backbone linkage lies in the disallowed region of the Ramachandran map. Figure
Figure 3. (A) and (B), Two orthogonal views of the model structure of the pig [alpha]3-GalT nucleotide-binding domain represented using the MOLSCRIPT program (Kraulis, 1991). Numbering of the secondary structure elements follows the [beta]-GlcT crystal structure. The UDP-Gal is represented using ball and stick and the Mg2+ ion by a sphere. (C) Graphical representation of UDP-Gal in the binding site of the model domain of pig [alpha]3-GalT. The yellow lines represent coordination contacts (d < 2.5 Å) around the Mg2+ cation. Hydrogen bonds are displayed by green dotted lines. Predicted interactions of [alpha]3-GalT with UDP-Gal
The structural equivalent of the [beta]-GlucT nucleotide binding site is an elongated pocket with [alpha]10 and [beta]8 on each side, [beta]11 and [beta]12 at the rear, and the loops between [beta]10 and [alpha]9 and [beta]11 and [alpha]10 at the bottom and top, respectively (Figure
When analyzing the sequence conservation among all eukaryotic [alpha]3-Gal or [alpha]-GalNAc transferases, it is notable that Glu308, the amino acid that is hypothesized to be involved in ribose binding is conserved among all members of this family. In terms of Mg2+ binding, Asp218 is also conserved, whereas Glu304 shows more variation. Asn224 is modeled to bind the [beta]-phosphate of UDP-Gal, and in the family is generally replaced by a basic amino acid (His or Lys) that can play the same role. Ile274, which is proposed to bind the pyrimidine moiety of UDP-Gal, is often replaced by another hydrophobic amino acids, that would not disrupt the hydrophobic character of the extended loop between [beta]10 and [alpha]9. Among the amino acids that are proposed to be involved in galactose binding, Ile301 and Glu302 are always conserved (with the exception of a Glu to Met mutation in the dog Forssman synthase) while Asn223 is often replaced by an aspartic acid that still hydrogen bonds with the O2 of the galactose. The model proposed for the [alpha]3-GalT C-terminal domain is therefore consistent for all other sequences of the eukaryotic [alpha]3-GalTs and blood group transferase protein family.
One intriguing question is why do the Leu266Met and Gly268Ala mutations alter the specificity from blood group A to blood group B, i.e., from a GalNAc to a Gal-transferase. In the present model, these two amino acids are in the loops between [beta]10 and [alpha]9, and are therefore close to the uridine base, whereas one might expect that they should directly interact with the sugar moiety, thus providing sugar specificity. However, the change in affinity between the two substrates appears to be due to a difference in kcat rather than in Km (Seto et al., 1997). Furthermore, Leu266 is not absolutely required for blood group A activity since a Ala amino acid is present at this position in the pig blood group A enzyme. Thus, the difference in enzymatic activity and substrate specificity may be due to a difference in the conformational properties of this particular loop, rather than a direct binding effect.
The amino acid sequences of the family of glycosyltransferases studied here, i.e., eukaryotic [alpha]3-GalTs and [alpha]3-GalNAcTs, have been previously compared to those of other classes of galactosyltransferases. A local similarity with eukaryotic [beta]4-GalTs first gave rise to the so-called DKKND motif (Joziasse et al., 1989). More recently, using the HCA method, three regions of sequence similarities were identified and labeled I to III (Breton et al., 1998). Region I contains a conserved DVDxxxxD/N motif. From the fold recognition study, this region corresponds to the loop that connects the N-terminal and C-terminal domain and extends in the [beta]8 strand and following loop. The conserved DVDxxxxD/N motif is located at the end of the [beta]8 strand, and the third residue (D) corresponds therefore to Asp218 that is proposed to interact with Mg2+ in the present model. The D/N corresponds to Asn223 that hydrogen bonds to the oxygen O2 of galactose. Region II of conserved amino acids corresponds to the loop between [beta]10 and [alpha]9. This loop ends up with the Ile residue involved in uridine binding. This region also contains the two amino acids which differs between the A and B transferases which have been suggested to be involved in nucleotide-sugar specificity (Yamamoto and Hakomori, 1990; Seto et al., 1997). Finally, the third conserved region from the sequence comparison study, i.e., region III, corresponds to [alpha]10 and the preceding loop. It appears, therefore, that the three regions which have been demonstrated to be conserved among eukaryotic [alpha]3-GalTs and [beta]4-GalTs correspond to the side, top, and bottom of the UDP-sugar binding site.
The DVD motif at the end of the [beta]8 strand seems to be of particular interest. In all families of bacterial and animal galactosyltransferases but one, this particular DxD motif is located just after a cluster of hydrophobic amino acids (Breton et al., 1998) that would correspond to a [beta]-strand according to HCA. Recently, this motif has been identified in other glycosyltransferases such as mannosyltransferases of which all use nucleoside diphosphate sugars and require divalent cations (Wiggins and Munro, 1998). Using photolabeling method, it has been demonstrated that mutations in the DXD motif in the large clostridial cytotoxins abolish the binding of nucleotide-sugar (Busch et al., 1998). These experimental data are in agreement with the present model since we propose that the second Asp residue of this motif is essential for binding the divalent cation associated with the nucleotide binding site. In the present study, a Mg2+ cation was included in the model, since this divalent cation is needed for [beta]-GlcT activity (Josse and Kornberg, 1962), but it should be noted that [alpha]3-GalT displays maximum activity in the presence of Mn2+ cation (Van den Eijnden et al., 1985).
Both [beta]-GlcT and glycogen phosphorylases catalyze the phosphate-dependent formation or breakdown of a O-glycosidic linkage. Their fold similarity could either be due to convergent evolution, or to a very remote evolutionary relationship. This latter hypothesis was concluded (Artymiuk et al., 1995; Holm and Sanders, 1995) based on the strong structural resemblance. However, the missing link in the evolutionary model could not be determined. Holm and Sanders (1995) searched for such candidates among glycosyltransferases responsible for glycogen or starch synthesis but could not find sequences with compatible secondary structure prediction. From the present study, we can postulate that eukaryotic [alpha]3-galactosyltransferases belongs to this superfamily. A preliminary report on fucosyltransferases (Breton et al., 1996) indicated that these enzymes could also adopt the [beta]-GlcT fold. Our current hypothesis is that most of the eukaryotic glycosyltransferases, as well as many bacterial ones, display a similar two-domains assembly, one being a Rossman type nucleotide binding domain. Sequence alignment
Pairwise alignments of [alpha]3-GalT and blood group transferase sequences were performed with the ALIGN program from the FASTA package (Myers and Miller, 1988). Multiple alignment of amino acid sequences was performed using the ClustalW method (Thompson et al., 1994). Secondary structure prediction
Secondary structure predictions were performed using three programs available on WWW servers. (1) The PHDsec method (Rost and Sander, 1993, 1994; http://www.EMBL-Heidelberg.DE/Services/index.html) uses a system of neural networks scheme that extracts conservation weights from a multiple sequence alignment. (2) PREDATOR (Frishman and Argos, 1997; http://www.embl-heidelberg.de/argos/predator/predator_info.html) takes as input a single protein sequence but also uses information from a set of related sequences. (3) The NPS method (http://pbil.ibcp.fr/NPSA/npsa_prediction.html) provides the consensus secondary prediction for one sequence using a set of different algorithms. The secondary structure predictions were run using either the pig [alpha]3-GalT sequence or the multiple alignment obtained by ClustalW. Fold prediction
The ProFIT program (Flöckner et al., 1995) from the ProCyon package (King's Beech, Biosoftware Solution, http://www.horus.com/sippl/) was used for fold recognition calculations using all [alpha]3-GalT sequences. The cytoplasmic tail, transmembrane domain, and stem were not included in the query sequence. Several other fold recognition programs were also tested such as TOPITS (Rost, 1995), THREADER2 (Miller et al., 1996), and FORESST (Di Francesco et al., 1997). Homology modeling
The pig [alpha]3-GalT C-terminal region sequence (160 amino acids) was aligned on the C-terminal domain of [beta]-GlcT, and the corresponding region of glycogen and maltodextrin phosphorylases with the help of the HCA program (Gaboriaud et al., 1987). The hydrophobic clusters were aligned visually, while also taking into account the secondary structure prediction. A library of four crystal structures was prepared containing the [beta]-GlcT (Morera and Freemont, personal communication), rabbit and yeast glycogen phosphorylases (PDB codes 1GPB and 1YHP), and E.coli maltodextrin phosphorylase (code 1AHP) from the Brookhaven protein databank (Abola et al., 1997). The pig [alpha]3-GalT sequence, together with the library of four crystal structures, served as input for the COMPOSER program (Blundell et al., 1988) in the Sybyl package (Tripos). Structurally conserved regions (SCRs) shared by the four crystal structures of the library were defined to correspond, where possible, to the secondary structure elements of [beta]-GlcT. All of the eight loops were modeled by using the most similar fragments in the library of four structures. Each one was submitted to local geometry optimization to release the steric conflicts. The resulting model was then screened using the PROCHECK program (Laskowski et al., 1993), and backbone linkages lying outside the allowed regions of the Ramachandran map were further optimized. Hydrogen were added on all atoms, and partial atomic charges were derived using the Pullman procedure. Docking of UDP-Gal
Before final optimization of the model, UDP-galactose was docked in the binding site. The UDP moiety was given the location observed in the crystal structure of [beta]-GlcT and the galactose residue was linked and oriented in the only conformation that does not yield to severe steric conflicts. Atom types and energy parameters available for carbohydrates (Pérez et al., 1995) within the TRIPOS force-field (Clark et al., 1989) were used, together with new parameters developed for the sugar-pyrophosphate linkage (Imberty and Petrova, unpublished results). A Mg2+ cation was located between the pyrophosphate group and the protein surface. The optimization of the complex was then run in successive cycles.
This work was supported by the following grants: Programme Physique et Chimie du Vivant-CNRS, Immunology Concerted Action 3026PL950004 and Xenotransplantation Project BIO4CT972242 of the BIOTECH program from European Union. C.B. is a staff member of Institut National de la Recherche Agronomique.
GalT, galactosyltransferase; GlcT, glucosyltransferase; GP, glycogen phosphorylase; Gal, galactose; Glc, glucose; GalNAc, N-acetylgalactosamine; UDP, uridine diphosphate.
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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