Bovine alpha1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases
2001; Springer Nature; Volume: 20; Issue: 4 Linguagem: Inglês
10.1093/emboj/20.4.638
ISSN1460-2075
AutoresLouis N. Gastinel, Christophe Bignon, A. Misra, Ole Hindsgaul, Joël H. Shaper, David H. Joziasse,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoArticle15 February 2001free access Bovine α1,3-galactosyltransferase catalytic domain structure and its relationship with ABO histo-blood group and glycosphingolipid glycosyltransferases Louis N. Gastinel Corresponding Author Louis N. Gastinel Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France Search for more papers by this author Christophe Bignon Christophe Bignon Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France Search for more papers by this author Anup K. Misra Anup K. Misra Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada The Burnham Institute, La Jolla, CA, 92037 Canada Search for more papers by this author Ole Hindsgaul Ole Hindsgaul Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada The Burnham Institute, La Jolla, CA, 92037 Canada Search for more papers by this author Joel H. Shaper Joel H. Shaper The Cell Structure and Function Laboratory, The Oncology Center and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, 21231-1000 USA Search for more papers by this author David H. Joziasse David H. Joziasse Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Search for more papers by this author Louis N. Gastinel Corresponding Author Louis N. Gastinel Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France Search for more papers by this author Christophe Bignon Christophe Bignon Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France Search for more papers by this author Anup K. Misra Anup K. Misra Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada The Burnham Institute, La Jolla, CA, 92037 Canada Search for more papers by this author Ole Hindsgaul Ole Hindsgaul Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada The Burnham Institute, La Jolla, CA, 92037 Canada Search for more papers by this author Joel H. Shaper Joel H. Shaper The Cell Structure and Function Laboratory, The Oncology Center and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, 21231-1000 USA Search for more papers by this author David H. Joziasse David H. Joziasse Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Search for more papers by this author Author Information Louis N. Gastinel 1, Christophe Bignon1, Anup K. Misra2,3, Ole Hindsgaul2,3, Joel H. Shaper4 and David H. Joziasse5 1Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 Chemin Joseph Aiguier, 13402 Marseille, Cedex 20, France 2Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2 Canada 3The Burnham Institute, La Jolla, CA, 92037 Canada 4The Cell Structure and Function Laboratory, The Oncology Center and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, 21231-1000 USA 5Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:638-649https://doi.org/10.1093/emboj/20.4.638 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info α1,3-galactosyltransferase (α3GalT, EC 2.4.1.151) is a Golgi-resident, type II transmembrane protein that transfers galactose from UDP-α-galactose to the terminal N-acetyllactosamine unit of glycoconjugate glycans, producing the Galα1,3Galβ1,4GlcNAc oligosaccharide structure present in most mammalian glycoproteins. Unlike most other mammals, humans and Old World primates do not possess α3GalT activity, which is relevant for the hyperacute rejection observed in pig-to-human xenotransplantation. The crystal structure of the catalytic domain of substrate-free bovine α3GalT, solved and refined to 2.3 Å resolution, has a globular shape with an α/β fold containing a narrow cleft on one face, and shares a UDP-binding domain (UBD) with the recently solved inverting glycosyltransferases. The substrate-bound complex, solved and refined to 2.5 Å, allows the description of residues interacting directly with UDP-galactose. These structural data suggest that the strictly conserved residue E317 is likely to be the catalytic nucleophile involved in galactose transfer with retention of anomeric configuration as accomplished by this enzyme. Moreover, the α3GalT structure helps to identify amino acid residues that determine the specificities of the highly homologous ABO histo-blood group and glycosphingolipid glycosyltransferases. Introduction Mammalian glycosyltransferases form a group of 100 or more enzymes that collectively participate in the biosynthesis of the glycans of glycoproteins, proteoglycans and glycolipids. α1,3-galactosyltransferase (α3GalT, EC 2.4.1.151) catalyzes the transfer of galactose (Gal) from UDP-Gal to glycoconjugate acceptors having LacNAc (Galβ1,4GlcNAc) as the non-reducing terminal disaccharide in the presence of Mn2+ as cofactor, according to the reaction: UDP-Gal + Galβ1,4GlcNAc-R → Galα1, 3Galβ1,4GlcNAc-R + UDP in which R may be a glycoprotein or a glycolipid (Blanken and Van den Eijnden, 1985). Both α3GalT and its enzymatic product, the Galα1,3Gal glycan structure, are expressed by New World primates (platyrrhines) and many non-primate mammals, but are absent from the tissues of Old World primates (catarrhines), including Homo sapiens (Galili et al., 1988). The molecular basis for the species-specific absence of the enzyme involves the inactivation of the locus encoding the α3GalT gene in the primate taxa that do not express the Galα1,3Gal epitope (Larsen et al., 1990; Joziasse et al., 1991; Joziasse and Oriol, 1999). A cDNA encoding α3GalT was first isolated from a bovine cDNA library (Joziasse et al., 1989). Other mammalian α3GalT orthologs have been cloned from mouse (Larsen et al., 1989; Joziasse et al., 1992), marmoset (Henion et al., 1994) and pig (Strahan et al., 1995). The α3GalT cDNA sequence predicts a type II transmembrane protein showing a structural domain organization similar to that of the other mammalian glycosyltransferases (Joziasse, 1992). The bovine enzyme contains a six amino acid N-terminal cytoplasmic tail linked to a single transmembrane domain (16 amino acids), which is connected by the 'stem region' to the lumenal, C-terminal catalytic domain. The stem region, rich in proline, glycine and polar/charged amino acids, is not conserved among α3GalT family members. N- and C-terminal truncations of recombinant, soluble α3GalT indicated that the bovine catalytic domain encompasses amino acids 87–368 (Henion et al., 1994). The amino acid sequence E80-V368 of α3GalT shares a large degree of homology with α1,3-glycosyltransferases responsible for the synthesis of Forssman and iso-globoside glycosphingolipids (44–50% identity; Haslam et al., 1996; Xu et al., 1999; Keusch et al., 2000) and the ABO histo-blood group antigens (45% identity; Yamamoto and Hakomori, 1990; reviewed in Hakomori, 1999), suggesting a common phylogenetic origin. Moreover, the human ABO and Forssman glycosyltransferase genes share the same chromosomal localization (chromosome 9q34) with the human α3GalT homolog HGT-10, and the genomic organization with murine α3GalT, which demonstrates a close evolutionary relationship (Joziasse et al., 1992; Yamamoto et al., 1995; Xu et al., 1999). Together, the various genes constitute an α1,3-glycosyltransferase gene family, which probably arose from a series of gene duplications. Subsequent divergence has produced enzymes that use different donor and acceptor substrates from those of bovine α3GalT (Table I). The high degrees of identity present all along the catalytic domain of the α1,3-glycosyltransferases suggest a common fold. The fact that catalytic properties have diverged makes this gene family a useful system for analyzing the role of the various amino acids of the catalytic domain in determining substrate preference. Table 1. Donor and acceptor substrate preference of the α1,3-glycosyltransferase family Donor substrate Acceptor substrate α3GalT UDP-Gal Galβ1,4GlcNAc-R A transferase UDP-GalNAc Fucα1,2Galβ1,3/4-R B transferase UDP-Gal Fucα1,2Galβ1,3/4-R Forssman synthase UDP-GalNAc GalNAcβ1,3Galα1,4Galβ1,4Glcβ1-ceramide Iso-globoside synthase UDP-Gal Galβ1,4Glcβ1-ceramide All enzymes produce α1,3-glycosyl linkages. The acceptor sugar is indicated in bold. The elucidation of the mechanism of enzymatic glycosyl transfer has been another impetus to our efforts to derive the three-dimensional structure of α3GalT. α3GalT transfers galactose from the donor UDP-α-D-Gal to the acceptor Galβ1,4GlcNAc-R, while retaining the galactose in the α-anomeric configuration. Earlier, crystal structures were derived for β4GalT1 (Gastinel et al., 1999), glucuronyltransferase I (Pedersen et al., 2000) and GlcNAc-transferase I (Unligil et al., 2000), all of which transfer a sugar via an inverting mechanism. Comparison of the catalytic center of these enzymes with that of α3GalT may produce insight into the mechanism that determines whether inversion or retention occurs. The α1,3GalT enzyme has recently attracted considerable attention because it synthesizes the Galα1,3Gal epitope. Naturally occurring anti-Galα1,3Gal antibodies in human serum (Galili et al., 1987) present a major barrier to the use of porcine and other non-primate organs for xenotransplantation in humans. Antibody binding to the Galα1,3Gal epitopes present on the vascular endothelium of the xenotransplants produces hyperacute graft rejection. Efforts are now in progress to overcome this difficulty by modifying the donor animal, the pig (reviewed in Cooper, 1998; Joziasse and Oriol, 1999). One strategy to this end could be the pre-treatment of pigs using an α3GalT-specific enzyme inhibitor. The structure-based design of such a drug will benefit from a knowledge of the three-dimensional structure of α3GalT. Here, we report on the crystal structure of the bovine α3GalT catalytic domain in both the absence and presence of UDP-Gal. This is the first described structure for a 'retaining' glycosyltransferase. The crystal structure reveals a globular α/β fold, and allows the description of the donor-binding site at atomic resolution. The structure also suggests amino acids involved in the acceptor-binding site. The amino acids responsible for the substrate specificity of strongly related enzymes such as the ABO histo-blood group glycosyltransferases and certain glycosphingolipid synthases are identified. Moreover, a hypothesis is proposed to explain the glycosyl transfer mechanism by retention of anomeric configuration. Results and discussion Overall protein structure Truncated bovine α3GalT (residues 80–368) produced as a seleniated molecule was crystallized and solved by the multiwavelength anomalous diffraction (MAD) phasing method using data sets collected at three wavelengths (Hendrickson et al., 1990) (Table II). The seleniated structure was refined to 2.8 Å and the seleniated model was used to refine the structural parameters against the 2.3 Å data collected from the native substrate-free α3GalT crystals (Table II). The substrate-bound α3GalT, obtained by soaking α3GalT crystals in the presence of Hg-UDP-Gal and Mn2+, was solved to 2.5 Å resolution using the refined native coordinates of the substrate-free α3GalT structure. Table 2. Crystallographic data and refinement statistics Sel-α3GalT Sel-α3GalT Sel-α3GalT α3GalT α3GalT + UDPG Wavelength (Å) 0.9796 0.9800 0.9324 0.9324 0.9324 Peak (W1) inflection (W2) remote (W3) Energy (Kev) 12.6566 12.6515 13.2971 13.2971 13.271 Resolution (Å) 30.0–2.8 30.0–2.8 30.0–2.8 30.0–2.0 30.0–2.5 Unit cell, a = b, c (Å) 95.55, 112.71 95.55, 112.71 95.55, 112.71 95.56, 112.71 95.6, 110.72 No. of unique reflections 10 366 10 366 10 366 31 286 18 010 [I/σ(I)] 5.7 (1.8) 5.6 (2.0) 7.2 (1.7) 7.5 (1.8) 9.3 (2.4) Rsym (%)a 7.9 (20) 7.7 (19.5) 7.5 (12) 5.8 (38.3) 4.3 (22.7) Ranomal (%) 8.0 (15.5) 5.9 (13.2) 6.7 (9.5) Completeness (%) 97.6 (97.6) 98.2 (98.2) 95.7 (95.7) 96.5 (96) 99.5 (99.5) Anomalous completeness (%) 84.2 (85) 88 (87) 80.3 (79.2) Multiplicity 3.4 (3.4) 3.7 (3.6) 3.5 (3.4) 5.6 (2.3) 6.7 (6.3) Resolution for the refinement (Å) 15.0–2.8 15.0–2.3 15.0–2.5 Rcryst (%)/Rfree (%)b 23/27 21/25 22/27 R.m.s.d. (bonds) (Å)/(angles (°) 0.0095/1.52 0.016/1.8 0.015/1.8 No. of atoms protein/water 2308/43 2393/130 2308/127 cofactorsc 20/1 36/1/1/11 Average B-factor (Å2) protein/water 53.5/60 50/50 66/68 cofactorsc 41/44 59/78/77/60 No. of φ/ψ angles (%) most favoured/allowed 80/16 86/12.8 87.1/12.5 The values in parentheses refer to data in the high resolution shell. a Rsym = Σhkl Σi|Ii − (I)|/Σ(I) b Rcryst = Σ(||Fp(obs)| − |Fp(calc)||/Σ|Fp(obs)| and Rfree = R-factor for a randomly selected subset (9.5%) of data that were not used to minimize the crystallographic residual. c Cofactors: UMP/Mn in the case of α3GalT and UDP-Gal/-Hg/Mn/galactose bound to E317 in the case of α3GalT + UDPG. The substrate-free α3GalT (Ser81-Asn367) and the substrate-bound α3GalT (Lys82-Thr358) form a globular protein with overall dimensions of 50 × 43 × 58 Å3 (Figure 1). The structure consists of 10 β-strands, six α-helices and six 310 helices, based on the PROMOTIF program (Hutchinson et al., 1996). The folding of the protein is that of an α/β protein with a central mixed twisted β-sheet of eight β-strands surrounded by four long α-helices. The structure starts at Ser81 with a short N-terminal α-helix (α1), followed by a β-hairpin containing one β-strand (β1), and then by a long connecting α-helix (α2). The central β-sheet can be divided into two portions. The first portion runs from Val129 to Met224, defining an N-terminal subdomain. It contains a β-sheet formed by four parallel β-strands in the strand order 4, 3, 2 and 5, surrounded by two long α-helices (α3 and α4). This N-terminal subdomain accounts for the binding of the nucleotide moiety of the nucleotide-sugar donor substrate because of the presence of unambiguous electronic density signatures of UMP and UDP-Gal occurring in the substrate-free and substrate-bound α3GalT crystals, respectively (Figures 1 and 2). The UMP molecule results from the elution step in the final affinity purification procedure of the enzyme using UDP-hexanolamine-Sepharose resin. The second portion of the central β-sheet consists of two parallel β-strands (β7 and β9) flanked by two antiparallel β-strands (β8 and β1) and with two long α-helices (α5 and α6) on one side. A second small β-sheet running almost parallel to the central β-sheet consists of two short antiparallel β-strands (β6 and β10). A structural homology search using the entire α3GalT structure and the DALI database revealed that its overall fold is unique (Holm and Sander, 1983). Figure 1.Overall view of the bovine substrate-bound α3GalT catalytic domain structure. Ribbon diagram of the molecule viewed down the open pocket. The three cysteine residues Cys223, Cys298 and Cys338 are shown in ball-and-stick form in yellow. The only Asn293 potentially available for N-glycosylation is shown in ball-and-stick form in red. N and C indicate the N- and C-termini of the molecule. Secondary structure elements are color coded as follows: α-helices in cyan, 310 helices in blue, the β-strands of the central twisted eight-stranded β-sheet in green, and the β-strands of the small two-stranded antiparallel β-sheet in magenta. Hg-UDP-Gal is shown in stick form color coded according to the nature of the atom. Download figure Download PowerPoint Figure 2.Close-up stereoview of the α3GalT UDP-Gal-binding site. (A) Hg-UDP-Gal is shown in ball-and-stick form and color coded depending on the nature of the atoms; the Mn2+ ion is shown as a pink sphere. Amino acid side chains interacting with Hg-UDP-Gal are shown in ball-and-stick form in yellow. The acidic residues from the motifs D225VD227 and the D316E317 are shown in ball-and-stick form in red. The four amino acid side chains of α3GalT residues at positions equivalent to the residues distinguishing human A-GT from B-GT are shown in ball-and-stick form in blue. (B) Stereoview of the electron density map (2Fo - Fc, 1σ) of the Hg-UDP-Gal-binding site. Download figure Download PowerPoint Strand β10 is followed by a loop including the C-terminal residues Thr358-Asn367, which completes the substrate-free structure. It has been shown previously that removing the last three residues, K374NV, of marmoset α3GalT inactivates the enzyme (Henion et al., 1994). The substrate-free α3GalT structure, in contrast to the substrate-bound structure, shows electron density for the C-terminal segment containing residues Lys359-Asn367. This segment is stabilized by hydrophobic interactions involving mainly residues V363, V364 and the tryptophans W249, W250 and W314. The absence of visible electron density for the C-terminal segment in the substrate-bound structure is not clearly understood. This region of the protein is presumed to be disordered in the substrate-bound crystal, being exposed to a large solvent channel, and is not involved in crystal contacts. None of the three cysteines of α3GalT bovine catalytic domain, C223, C298 and C338, is engaged in disulfide bridges (Figure 1), which is consistent with a recent analysis of bovine α3GalT structure using liquid chromatography and electrospray ionization-tandem mass spectrometry (Yen et al., 2000). The distances between the two strictly conserved cysteines, C223 and C298, is 13 Å. Each of the two conserved cysteine residues is located in a cluster of hydrophobic residues enclosed within the central part of the α3GalT protein. In this central position, changes are likely to affect protein fold integrity, which could explain their strict conservation among all the α1,3-glycosyltransferase family members. The packing of α3GalT crystals shows a close interaction between two symmetrical α3GalT molecules mediated by their respective N-terminal subdomains rotated over ∼90°. This hydrophobic interaction involves mainly residues V170, P174 and L175 from the long connecting loop between β3 and β4, and residues F184, V186 and F187 from strand β4. The total buried surface represents 840 Å2 (calculated with TURBO software; A.Roussel and C.Cambillau, personal communication). So far, dimerization of soluble α3GalT has not been reported; it has been proposed that different glycosyltransferases may form non-covalent oligomers during Golgi sorting/trafficking. It is conceivable that the homologous UDP-binding subdomain shared by Golgi glycosyltransferases of different specificities (see below) might support their non-covalent association, increasing their efficiency in glycan biosynthesis. α3GalT catalytic pocket The catalytic pocket of α3GalT was identified by the presence of: (i) a narrow cleft measuring ∼14 × 14 Å on its molecular surface, formed between the N-terminal subdomain and the C-terminal portion of the protein; (ii) a large surface portion where amino acid residues that are invariant within the α1,3-glycosyltransferase family are concentrated; and (iii) one UMP molecule found in the substrate-free, or one Hg-UDP-Gal molecule found in the respective substrate-bound structures (Figures 1 and 2). The catalytic pocket contains the solvent-exposed sequence D225VD227, which was identified as the DXD motif, known to be well conserved in many mammalian glycosyltransferases (Wiggins et al., 1998). The α3GalT pocket is made up of 19 solvent-exposed residues, two hydrophobic, 12 polar (six tryptophan residues) and five ionizable side chain residues. The bottom of the cavity is formed from the β-strand β8 containing residues H280, A281 and A282, and its sides are lined with the following residues: Y139, W195, S199, R202, D225, D227, Q228, Q247, W249, W250, T259, W314, D316, E317 and W356 (Figure 2). Eight of these residues, Y139, W195, S199, R202, D225, D227, W314 and E317, are invariant in the α1,3-glycosyltransferase family. They are involved in the binding of the UDP moiety of the UDP-sugar donor substrate and may contain the acidic residue responsible for the nucleophilic attack on the C1 atom of the transferred sugar. The other residues, Q228, Q247, W249, W250, T259, H280, A281, A282, D316 and W356, are conserved only in all α3GalT orthologs but are different in A-GT, B-GT, iGB3 synthase and Forssman glycosyltransferase. This list of residues might include some of the amino acids that interact with the donor substrate sugar (galactose or N-acetylgalactosamine) and with the acceptor substrate. UDP-galactose-binding site Hg-UDP-Gal binds across the depression found on the α3GalT molecular surface, with its uridine and ribose moieties maintained by conserved residues that form a small pocket in the N-terminal subdomain. The uracil base binds via aromatic stacking involving Y139, W195 and I198, and its N3 and O2 atoms hydrogen-bond to V136 (Table III; Figure 2A). The ribose O3′ forms water-mediated and direct hydrogen bonds with the NH1 of R202 and the N atom of V226. Table 3. Interatomic distances between Hg-UDP-Gal, Mn2+, bound water molecules, UMP and α3GalT protein atoms in substrate-free and substrate-bound structures Substrate-free structure Substrate-bound structure Interacting atoms Distance (Å) Interacting atoms Distance (Å) Uracil N3 V136 O 3.0 Uracil N3 V136 O 3.0 Uracil O2 V136 O 3.0 Uracil O2 V136 N 2.9 Uracil O2 V136 N 2.9 Ribose O2′ F134 O 3.0 Uracil O4 R138 NH2 2.7 Ribose O3′ R202 NH1 2.7 Ribose O2′ V226 N 2.7 Ribose O3′ Wat 89 2.5 Ribose O2′ Wat 2 2.7 Ribose O3′ V226 N 3.4 Ribose O3′ D227 N 3.3 O2Pα Y139 OH 2.6 O3Pα Y139 OH 2.7 Mn2+ D225 OD2 2.5 Mn2+ D225 OD2 2.6 Mn2+ D227 OD1 2.4 Mn2+ D227 OD2 2.7 Mn2+ D227 OD2 2.4 Mn2+ D227 OD1 2.4 Mn2+ Wat 82 2.9 Mn2+ Wat 97 2.3 Mn2+ O2Pβ 2.9 Mn2+ O1Pα 2.1 Mn2+ O3Pα 2.5 Gal O2 A282 N 3.2 Gal O3 Wat 84 3.2 Gal O3 A281 O 3.1 Gal O3 D225 OD1 3.2 Gal O3 D225 OD2 3.4 Gal O3 R202 NH2 2.9 Gal O4 D316 OD1 2.9 Gal O4 D316 OD2 2.8 Gal O4 E317 N 3.4 Hg Y139 OH 2.5 The galactose moiety is involved in several interactions with the protein, in particular with R202, D225, A281, A282, D316 and E317 (Table III; Figure 2). An Mn2+ atom is found in an approximately octahedral coordination state in which two of the coordination atoms, O3Pα and O2Pβ, are from the α- and β-phosphate of the UDP molecule (Table III). D227 forms a bidentate interaction through OD1 and OD2, and D225 interacts through OD2. The last ligand is from a water molecule. Three of the six Mn2+ coordination sites are from direct interaction with α3GalT protein, which might produce substantial affinity of the protein for Mn2+ even in the absence of donor substrate. In the GnT I and GlcAT-1 structures, only one and two direct interactions, respectively, have been observed between Mn2+ and protein, which excludes the existence of an independent metal-binding site in these enzymes. Only a few significant differences are observed between the binding of UMP in the substrate-free structure and that of Hg-UDP-Gal in the substrate-bound structure (Table III). In the case of the substrate-free structure, the uracil N3, O2 and O4 atoms hydrogen-bond to V136 and the R138 NH2 atom, respectively. The two ribose oxygens O2′ and O3′ form one water-mediated and two direct hydrogen bonds to V226 and D227 main chain nitrogen atoms. These differences may be due to the presence of the mercury atom bound to the C5 atom of the uracil ring. Five of the six possible coordination sites of Mn2+ are used. One coordination atom, O1Pα, is from the UMP molecule; the other four coordination atoms are equivalent to those in the substrate-bound structure (Table III). The α1,3-glycosyltransferase family: donor substrate specificity The glycosyltransferases responsible for the synthesis of ABO histo-blood group carbohydrates and certain glycosphingolipids belong to the same α1,3-glycosyltransferase family as bovine α3GalT, because they show a high level of identity in the catalytic domain (Figure 3). The human blood group transferases A-GT and B-GT, which transfer N-acetylgalactosamine and galactose, respectively, to α1,2-fucosylated complex-type glycans, share strict amino acid sequence identity except at four positions, from A-GT to B-GT: Arg146→Gly146, Gly235→Ser235, Leu266→ Met266 and Gly268→Ala268. Chimeric enzymes have been constructed at these positions between A-GT and B-GT, and only two positions, 266 (Leu→Met) and 268 (Gly→Ala), were found to be crucial for defining the donor preference, GalNAc versus Gal (Seto et al., 1999). Figure 3.Sequence alignment of bovine α3GalT and homologous proteins belonging to the family of related α1,3-glycosyltransferases. The invariant residues are highlighted in red with a gray background, whereas conserved residues have an orange background. Cysteine residues are displayed in green. Secondary structure elements of bovine α3GalT are indicated beneath the sequences color coded as in Figure 1A. Amino acid sequences used for the alignment are: bovine α3GalT (P14769; Joziasse et al., 1989), pig_13GT (P50127; Strahan et al., 1995), marmoset_13GT (Q28855; Henion et al., 1994), mouse_13GT (P23336; Larsen et al., 1989), rat_iGb3 synthase (AF248543; Keusch et al., 2000), humB_GT and humA_GT (AF134414 and AF134412; Yamamoto et al., 1990), hum_forssman (DDBJ/EMBL/GenBank 163572; Xu et al., 1999) and dog_forssman (Q95158; Haslam et al., 1996). Residues exposed at the surface in the pocket of the substrate-bound α3GalT structure are indicated by closed black circles. Residues that interact with the UDP portion of Hg-UDP-Gal are indicated by closed red circles. The four residues that differentiate between the humA_GT and humB_GT sequences are indicated by closed blue triangles. The first well defined residue in the electron density map of the substrate-bound α3GalT structure, Lys82, is indicated by a closed magenta triangle. The Asn293 involved in the only potential N-glycosylation site is indicated by a closed green triangle. The bovine α3GalT sequence is numbered every tenth residue. Download figure Download PowerPoint In the α3GalT structure, the two equivalent positions are His280 (Leu266 or Met) and Ala282 (Gly268 or Ala), respectively, both of which are located in the catalytic pocket of the enzyme. The residues His280 and Ala282 are located in β-strand β8 in the middle of the central β-sheet forming the bottom of the catalytic pocket (Figure 2A). His280 does not interact directly with galactose, but replacing it by either of the hydrophobic residues methionine (B-GT) or leucine (A-GT) will result in a local change in the shape or size of the cavity in proximity to the chemical group attached to the donor sugar C2 atom. The side chain of Ala282, which is replaced by glycine (Gly268) in the human A-GT, is pointing upward from the bottom of the catalytic cavity and could restrict by steric hindrance the access of the N-acetyl moiety of the UDP- N-acetylgalactosamine, the sugar donor of the human A-GT. A cDNA coding for the rare O2 allele of the ABO histo-blood group has been described recently as containing a point mutation whereby Ala268 is replaced by Arg268, resulting in the complete inactivation of the enzyme (Hakomori, 1999). In the bovine α3GalT structure, a long and charged arginine side chain, replacing the alanine residue at the bovine α1,3GalT equivalent position 282, should completely block the donor sugar-binding site by steric hindrance and electrostatic incompatibility. Acceptor specificity of α3GalT The members of the α1,3-glycosyltransferase family differ in the nature of the acceptor substrate that they use (Table I). In the absence of X-ray data on acceptor substrate bound to α3GalT crystals, we can only make assumptions as to the probable position of the acceptor substrate in the protein. Surface hydrophobic residues, particularly solvent-exposed tryptophans, have been suggested to interact with the sugar ring by hydrophobic stacking interactions. Among the five solvent-accessible tryptophan residues found in the α3GalT catalytic pocket (W195, W249, W250, W314 and W356), only two, W249 and W356, are specific to α1,3-glycosyltransferases that transfer galactose to non-fucosylated LacNAc acceptors. Trp249 of α3GalT is replaced by either serine or glycine in the A-GT, B-GT and Forssman synthases, whose acceptor substrate is either a β1,3/4-linked α1,2-fucosylated galactose or a β1,3-linked N-acetylgalactosamine (Table I). Trp356 of α3GalT is replaced by alanine (Ala343) in the ABO histo-blood group glycosyltransferases. This modification indicates that Ala343 might be close to the fucose-binding site of α1,2-fucosylated acceptors. A tryptophan residue replacing alanine at this
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