Fold recognition study of α3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain
Abstract and Figures
The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.
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... Recently, the structure of this enzyme was used as a template to predict the structure and fold of the pig ␣-1,3-GalT. 8 In the past few months, the X-ray structures of three glycosyltransferases, the bacterial SpsA, 9 for which the substrate specificity is undefined, the bacterial -1,4-galactosyltransferase T1 10 (4Gal T1), and the rabbit N-acetylglucosaminyltransferase I 11 (GnT I) have been solved. Comparison of enzyme structures revealed that although the topology of the UDP binding domain of -glucosyltransferase from bacteriophage T4 is similar to that of the other two enzymes, the mode of nucleotide binding is different. ...
... It is noteworthy that some of these residues, such as Tyr139, Asp-197, are conserved across various species. 8 Because no solution data are available on this system it is very difficult to assess the importance of these residues. As we have mentioned earlier, no attempt was made to alter the side-chain conformations of the protein during the docking experiments. ...
... In the crystal structure of the complex of SpsA with UDP, the UDP is bound at the active site of the enzyme. 8 The uracil ring of the bound UDP is placed into the cavity where its carbonyl and amide hydrogens form two hydrogen bonds with side chains of Arg-71 and Asp-39, respectively. Apart from these hydrogen bond interactions, a favorable stacking interaction between the uracil ring and side chain of Tyr-11 is possible. ...
A homology model of α-1,3-galactosyltransferase (α-1,3-GalT), the retaining enzyme responsible for the formation of α-galactosyl epitopes, has been developed by means of molecular modeling using the SpsA glycosyltransferase structure. A protein-ligand docking approach was used to model α-1,3-GalT complexed with UDP and UDP-Gal. The comparison of structural features found in the α-1,3-GalT homology model with available structural data on this class of enzymes revealed similarities in the UDP-binding pocket. In the predicted structure of the complexes, the pyrophosphate interacts with the DVD motif (Asp-225, Val-226, and Asp-227) of α-1,3-GalT through the Mn2+ cation. The uridine part of the UDP binds into the well-defined cavity that consists of Phe-134, Tyr-139, Ile-140, Val-136, Arg-194, Arg-202, Lys-209, Asp-173, His-218, and Thr-137 in a conformation that is generally observed in the crystal structures of other glycosyltransferase complexes. Proteins 2001;44:428–434. © 2001 Wiley-Liss, Inc.
... Ces différentes structures sont biosynthétisées grâce à l'intervention des N-acétylglucosaminyltransférases III à VI sur l'oligosaccharide GlcNAc2Man3GlcNAc2 (Brockhausen and Schachter, 1996). Sur ces différentes antennes vont pouvoir venir se greffer différents résidus comme du galactose qui peut être ajouté en β-1,4 aux GlcNAc distales, contrairement aux plantes où ce résidu est retrouvé lié en β-1,3 (Breton et al., 1998b;Imberty et al., 1999). Ces résidus galactose sont généralement modifiés par l'ajout d'acides sialique en α-2,3ou α-2,6 catalysé par des sialyltransférases (Audry et al., 2011;Choi et al., 2014;Jeanneau, 2004). ...
La N-glycosylation est un événement co- et post-traductionnel majeur de la synthèse protéique chez les eucaryotes. Actuellement, peu d’informations concernant ce processus sont disponibles chez les microalgues. Dans ces travaux, nous avons poursuivi l’exploration de la voie de N-glycosylation de la diatomée modèle P. tricornutum en focalisant spécifiquement notre travail sur la caractérisation de trois α1,3-fucosyltransférases putatives. Nos analyses ont permis de remettre en cause l’annotation pour deux des gènes codant pour ces fucosyltransférases et d’en proposer de nouvelles. Afin de réaliser l’étude de la localisation subcellulaire de ces glycosyltransférases, nous avons utilisé une stratégie de sur-expression de ces protéines fusionnées avec une étiquette. Les observations menées par des approches de microscopie confocale et de microscopie électronique à transmission ont permises de mettre en évidence la localisation subcellulaire golgienne de ces enzymes, ainsi que de trois autres glycosyltransférases utilisées comme témoin dans ce travail. Cette étude a permis de réaliser la première localisation subcellulaire de glycosyltransférases chez les microalgues. P. tricornutum est une diatomée pléïomorphique qui possède trois morphotypes majeurs différents appelés ovale, fusiforme et triradié. Le passage d’un morphotype à un autre dépend fortement des conditions environnementales. Cependant, peu d’informations sont disponibles concernant les mécanismes et la signification physiologique de cette morphogenèse. Afin d’apporter de nouvelles réponses, nous avons réalisé une analyse comparative du transcriptome des trois morphotypes par une approche transcriptomique à haut débit appelée RNASeq pour RNA-Sequencing. Enfin, en plus d’élargir les connaissances sur la morphogénèse de P. tricornutum, les données générées ont également permis d’identifier de nouveaux gènes de référence utilisables pour des analyses de qRT-PCR.
... Sur ces différentes antennes viennent se greffer divers résidus ou séquences oligosaccharides. Par exemple, un résidu galactose peut être ajouté en b-1,4 aux résidus GlcNAc distales sous l'action de la b-1,4-galactosyltransférase, ce qui diffère des plantes où le galactose est lié en b-1,3 Imberty et al., 1999). L'association de ces deux sucres forme un disaccharide appelé lactosamine ( figure 22). ...
La N-glycosylation est un événement co- et post-traductionnel majeur dans la synthèse protéique. Alors que ce processus est bien connu chez les eucaryotes supérieurs, peu d'informations sont disponibles chez les microalgues. Dans ce travail, nous décrivons la voie de N-glycosylation d'une diatomée modèle, Phaeodactylum tricornutum. L'analyse bio informatique de son génome a révélé la présence de séquences codant potentiellement pour, des transférases responsables de la synthèse du précurseur oligosaccharidique, des sous-unités du complexe oligosaccharyltransférase, des glucosidases du RE et des chaperonnes nécessaires au contrôle qualité des protéines, ainsi que pour une a-mannosidase I golgienne impliquée dans la démannosylation des N-glycannes oligomannosidiques. Des analyses structurales des N-glycannes de P. tricornutum ont démontré que leurs protéines endogènes portent principalement des N-glycannes oligomannosidiques possédant de 5 à 9 résidus mannose. Cependant, une population" mineure de N-glycannes plus matures a également été détectée comme le Manz FucGlcNẢc2. De plus, un gène codant potentiellement pour une N acétylglucosaminyltransférase I (GnT I), glycosyltransférase golgienne qui initie la formation des N-glycannes complexes, a été prédit dans le génome de cette diatomée. Nous avons démontré que ce gène code pour une GnT I fonctionnelle, capable de restaurer la maturation des N-glycannes complexes, dans des cellules mutantes d'ovaire de hamster chinois Lecl, déficientes en activité GnT I endogène. L'expression de ce gène chez P. tricornutum a été mise en évidence en fonction des conditions de culture, du morphotype et de la souche. L'ensemble de ces données suggèrent la présence d'une voie de N- glycosylation GNT I dépendante chez P. tricornutum
... They have been divided into over 93 families based on amino acid sequence similarities [14][15][16]. Although GTs exhibit considerable degree of diversity in families, they have been observed to be classified into only two different folds, GT-A and GT-B, based on 3-D structural analysis [17]. Remarkably, these two folds with no significant sequence identity have been shown to contain a similar active-site domain. ...
Glycosyltransferase (GT) catalyzes the transfer of a sugar moiety to acceptor substrates such as secondary metabolites. The majority of GTs has two structural folds, GT-A and GT-B based on 3-D structural analysis. The limited structural fold diversity is compensated by a highly divergent acceptor binding domain for conferring sufficient substrate promiscuity. Various GTs have been engineered to further enhance the glucose transfer activity and expand substrate promiscuity by error-prone PCR and site-directed mutagenesis. Engineered GT-catalyzed glycosylation will certainly play a key role in the generation of scaffold for new drug discovery and control of drug pharmacokinetics.
... The enzymes responsible for polysaccharide biosynthesis in plants can be classified into two groups, the polysaccharide synthases and the glycosyltransferases, which are in turn classified within the overall "glycosyl transferase" (GT) class of carbohydrate-modifying enzymes (Coutinho and Henrissat, 1999). The GTs are among the largest groups of enzymes known and have been divided into more than 70 families based on sequence similarities, the existence of certain motifs, hydrophobic cluster analysis (HCA) and their catalytic specificity (Campbell et al., 1997;Imberty et al., 1999;Ross et al., 2001;Rosen et al., 2004). Although there are a large number of glycosyltransferases in any plant, the biochemical activity of relatively few plant GTs has been demonstrated so far (Keegstra and Raikhel, 2001). ...
The cell walls of the starchy endosperm of barley are relatively minor components of the grain in comparison to starch and
protein, but exert considerable influence over many malting quality characteristics. Wall components such as (1,3;1,4)-β-D-gmcans, which are commonly but less precisely known as mixed linkage β-glucans, can have direct and often detrimental effects on various processes in the brewery and on the quality and shelf life
of the final beer. The same wall components can have indirect effects on other quality characteristics, such as malt extract
and fermentability. The undesirable nature of many of these direct and indirect effects has led breeders, maltsters and brewers
to develop precise assays to monitor levels of (1,3;1,4)-β-D-glucans in barley grain and malt, and in many cases to select against high levels of this polysaccharide. While commercial
interest in endosperm cell wall components has been focused on (1,3;1,4)-β-D-glucans, the walls contain other components that have similar chemical and physicochemical properties and which merit further
attention in the context of malting quality of barley. Here, we will examine the properties and roles of all components of
barley endosperm walls in malting quality and relate these to the biological functions of cell walls in grain development
and germination. There is a good deal of information available on the physiology and enzymology of wall degradation in germinated
barley grain, but only recently have we been able to identify enzymes involved in the biosynthesis of key wall constituents.
UDP-galactose:β-galactosyl-α1,3-galactosyltransferase (α3GT) catalyzes the synthesis of galactosyl-α-1,3-β-galactosyl structures
in mammalian glycoconjugates. In humans the gene for α3GT is inactivated, and its product, the α-Gal epitope, is the target
of a large fraction of natural antibodies. α3GT is a member of a family of metal-dependent-retaining glycosyltransferases
that includes the histo blood group A and B enzymes. Mn2+activates the catalytic domain of α3GT (α3GTcd), but the affinity reported for this ion is very low relative to physiological
levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn2+ binding to two sites. At physiological metal ion concentrations, Zn2+ gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was
perturbed by substituting Co2+and Zn2+ for Mn2+ and by mutagenesis of a conserved D149VD151 sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were
found to be essential for activity, and the kinetic properties of a Val150 to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose
and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase
activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition
state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar
bond cleavage.
The catalytic mechanism of the GlcNAc transfer by inverting N-acetylglucosaminyltransferases is explored for the first time using ab initio quantum chemical methods. The structural model describing the active site where the reaction occurs consists of all essential molecules or their fragments assumed to be involved in the mechanism: a complete sugar-donor molecule, UDP-GlcNAc, a hydroxyl group of the oligosaccharide-acceptor modeled by methanol, a divalent metal cofactor represented by Mg2+, as well as the essential parts of the catalytic acid (A) and catalytic base (B) modeled by acetic acid and acetate molecules. Different possible mechanisms of reaction have been followed by means of several two-dimensional potential energy maps calculated as a function of predefined reaction coordinates. Potential energy surfaces calculated at the HF/6-31G* level revealed 11 transition states and five intermediates associated with distinct possible reaction pathways. All stationary points, transition states, and intermediates, were characterized at HF/6-31G*, HF/6-31++G**//HF/6-31G*, DFT/B3LYP/6-31G*, and DFT/B3LYP/6-31++G**//DFT/B3LYP/6-31G* levels. A detailed description of the reaction pathways that includes energetic evaluations and the structural modifications of the different participants occurring along the catalytic process is given, followed by a discussion on their feasibility, consequences, and implications for the catalytic mechanism. Among the different reaction pathways, a stepwise reaction pathway assuming the enrolment of only a catalytic base appeared to be the most probable reaction path and consistent with the existing experimental data.
Glycosyl esters of nucleoside di or mono-phosphates, generally referred to as “sugar nucleotides”, serve as a sugar donors during the biosynthesis of oligo- and polysaccharides; they are therefore of a primary importance in carbohydrate metabolism in the living world. Molecular dynamics simulations were used to explore the conformational flexibility of one nucleotide sugar, UDP-glucose (UDP-Glc). The AMBER program package was used with some new parameters especially developed for nucleotide sugars. Several simulations on this molecule in aqueous solution, each of 2 ns duration, were carried out for increasing concentrations of monovalent K and divalent Mg ions. For the monovalent ion, it is revealed that its presence and concentration is crucial for the conformational behavior, resulting in the stabilization of the extended conformation. The preferred location of K is in close proximity to the negatively charged phosphate oxygens, but the ion moves freely and can occupy other sites. Since the size of this cation is close that of the water molecules, the hydration scheme is not perturbed. Completely different results are obtained when the divalent Mg cation is introduced in the simulation. A very strong interaction is established between the phosphate group and the cation; as a result the UDP-Glc molecule is locked in a rigid extended geometry. The analyses of the trajectories provide new insight on the role of the metal ion in the catalytic mechanism of glycosyltransferases.
Although the synthesis of cell wall polysaccharides is a critical process during plant cell growth and differentiation, many of the wall biosynthetic genes have not yet been identified. This review focuses on the synthesis of non-cellulosic matrix polysaccharides formed in the Golgi apparatus. Our consideration is limited to two types of plant cell wall biosynthetic enzymes: glycan synthases and glycosyltransferases. Classical means of identifying these enzymes and the genes that encode them rely on biochemical purification of enzyme activity to obtain amino acid sequence data that is then used to identify the corresponding gene. This type of approach is difficult, especially when acceptor substrates for activity assays are unavailable, as is the case for many enzymes. However, bioinformatics and functional genomics provide powerful alternative means of identifying and evaluating candidate genes. Database searches using various strategies and expression profiling can identify candidate genes. The involvement of these genes in wall biosynthesis can be evaluated using genetic, reverse genetic, biochemical, and heterologous expression methods. Recent advances using these methods are considered in this review.
The catalytic domain of bovine α1→3-galactosyltransferase (α3GalT), residues 80–368, have been cloned and expressed, in Escherichia coli. Using a sequential purification protocol involving a Ni2+ affinity column followed by a UDP-hexanolamine affinity column, we have obtained a pure and active protein from the soluble fraction which catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetyllactosamine (LacNAc) with a specific activity of 0.69 pmol/min/ng. The secondary structural content of α3GalT protein was analyzed by Fourier transform infrared (FTIR) spectroscopy, which shows that the enzyme has about 35% β-sheet and 22% α-helix. This predicted secondary structure content by FTIR spectroscopy was used in the protein sequence analysis algorithm, developed by the Biomolecular Engineering Research Center at Boston University and Tasc Inc., for the assignment of secondary structural elements to the amino acid sequence of α3GalT. The enzyme appears to have three major and three minor helices and five sheet-like structures. The studies on the acceptor substrate specificity of the enzyme, α3GalT, show that in addition to LacNAc, which is the natural substrate, the enzyme accepts various other disaccharides as substrates such as lactose and Gal derivatives, β-O-methylgalactose and β-D-thiogalactopyranoside, albeit with lower specific activities. There is an absolute requirement for Gal to be at the non-reducing end of the acceptor molecule which has to be β1→4-linked to a second residue that can be more diverse in structure. The kinetic parameters for four acceptor molecules were determined. Lactose binds and functions in a similar way as LacNAc. However, β-O-methylgalactose and Gal do not bind as tightly as LacNAc or lactose, as their Kia and KA values indicate, suggesting that the second monosaccharide is critical for holding the acceptor molecule in place. The 2′ and 4′ hydroxyl groups of the receiving Gal moiety are important in binding. Even though there is large structural variability associated with the second residue of the acceptor molecule, there are constraints which do not allow certain Gal-R sugars to be good acceptors for the enzyme. The β1→4-linked residue at the second position of the acceptor molecule is preferred, but the interactions between the enzyme and the second residue are likely to be non-specific.
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
Computer modeling has become a valuable component of studies of carbohydrate three-dimensional structures and their relationship to function and properties. In this paper we examine the methods required for conformational modeling of carbohydrates, and we present a series of tools that have been developed to this end. These tools can be integrated into three-dimensional real-time molecular modeling software. A data base of pre-optimized carbohydrate fragments has been established to be used further in the construction of much more complex molecules. In addition we describe some possible uses of a data base of three dimensional structures of the disaccharide fragments present in the glycan moiety ofN-glycoprotein. A molecular mechanical force field appropriate for the conformational analysis of oligosaccharides has been derived by the addition of new parameters to the Tripos force field and is compatible with protein simulations. The new parametrization has been assessed in three stages of increasing complexity: computations of potential energy surfaces, conformational refinement of relevant oligosaccharides, modeling at the atomic level of a protein/carbohydrate complex.
The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
The MOLSCRIPT program produces plots of protein structures using several different kinds of representations. Schematic drawings, simple wire models, ball-and-stick models, CPK models and text labels can be mixed freely. The schematic drawings are shaded to improve the illusion of three dimensionality. A number of parameters affecting various aspects of the objects drawn can be changed by the user. The output from the program is in PostScript format.
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A systematic technique for protein modelling that is applicable to the design of drugs, peptide vaccines and novel proteins is described. Our approach is knowledge-based, depending on the structures of homologous or analogous proteins and more generally on a relational data base of protein three-dimensional structures. The procedure simultaneously aligns the known tertiary structures, selects fragments from the structurally conserved regions on the basis of sequence homology, aligns these with the ‘average structure’ or ‘framework’, builds on the loops selected from homologous proteins or a wider database, substitutes sidechains and energy minimises the resultant model. Applications to modelling an homologous structure, tissue plasminogen activator on the basis of another serine proteinase, and to modelling an analogous protein, HIV viral proteinase on the basis of aspartic proteinases, are described. The converse problem of ab initio design is also addressed: this involves the selection of an amino acid sequence to give a particular tertiary structure, in this case a symmetrical domain of two Greek-key motifs.
A molecular mechanics force field implemented in the Sybyl program is described along with a statistical evaluation of its efficiency on a variety of compounds by analysis of internal coordinates and thermodynamic barriers. The goal of the force field is to provide good quality geometries and relative energies for a large variety of organic molecules by energy minimization. Performance in protein modeling was tested by minimizations starting from crystallographic coordinates for three cyclic hexapeptides in the crystal lattice with rms movements of 0.019 angstroms, 2.06 degrees, and 6.82 degrees for bond lengths, angles, and torsions, respectively, and an rms movement of 0.16 angstroms for heavy atoms. Isolated crambin was also analyzed with rms movements of 0.025 angstroms, 2.97 degrees, and 13.0 degrees for bond lengths, angles, and torsions respectively, and an rms movement of 0.42 angstroms for heavy atoms. Accuracy in calculating thermodynamic barriers was tested for 17 energy differences between conformers, 12 stereoisomers, and 15 torsional barriers. The rms errors were 0.8, 1.7, and 1.13 kcal/mol, respectively, for the three tests. Performance in general purpose applications was assessed by minimizing 76 diverse complex organic crystal structures, with and without randomization by coordinate truncation, with rms movements of 0.025 angstroms, 2.50 degrees, and 9.54 degrees for bond lengths, angles and torsions respectively, and an average rms movement of 0.192 angstroms for heavy atoms.
The possibility of defining effective potentials from known protein structures, which are sufficiently accurate to be used for protein-structure-prediction purposes, is investigated. Three types of distance potentials and three types of backbone torsion potentials are defined, based on propensities of amino acid pairs to be separated by a given spatial distance or to be associated to a backbone torsion angle domain. Their differences reside in the way the physical correlations between the amino acids and the conformational states are extracted from the bulk interactions due to the presence of many residues in a protein. For the distance potentials, a physical meaning can be associated to the different definitions, given that some of the potentials favor hydrophobic interactions and others favor interactions between oppositely charged residues. The performance of the different torsion and distance potentials in structure prediction procedures, in particular native-fold recognition and evaluation of protein stability changes upon point mutations, is analyzed. It appears to differ according to the specific proteins and protein environments. In particular, one of the distance potentials performs better than the others for membrane proteins and in protein regions involving charged residues, but less well in other protein regions. Furthermore, the dependence of the potentials on the characteristics of the proteins from which they are derived is analyzed. It is shown that the dependence of the potentials on the length, amino acid composition and secondary-structure content of the proteins from the dataset is either very limited or rather strong, according to the type of potential. The results obtained suggest that the main problem limiting the performance of database-derived potentials is their lack of universality : each potential describes with satisfactory accuracy only the interactions present in certain protein environments.
Human serum contains natural antibodies (NAb), which can bind to endothelial cell surface antigens of other mammals. This is believed to be the major initiating event in the process of hyperacute rejection of pig to primate xenografts. Recent work has implicated galoctosyl 1,3 galactosyl 1,4 N-acetyl-glucosaminyl carbohydrate epitopes, on the surface of pig endothelial cells as a major target of human natural antibodies. This epitope is made by a specific galactosyltransferase (1,3 GT) present in pigs but not in higher primates. We have now cloned and sequenced a full-length pig 1,3 GT cDNA. The predicted 371 amino acid protein sequence shares 85% and 76% identity with previously characterized cattle and mouse 1,3 GT protein sequences, respectively. By using fluorescence and isotopic in situ hybridization, the GGTA1 gene was mapped to the region q2.10–q2.11 of pig chromosome 1, providing further evidence of homology between the subterminal region of pig chromosome 1q and human chromosome 9q, which harbors the locus encoding the AB0 blood group system, as well as a human pseudogene homologous to the pig GGTA1 gene.