Bovine α1,3‐galactosyltransferase catalytic domain structure and its relationship with ABO histo‐blood group and glycosphingolipid glycosyltransferases

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.

[1]  S. Elliott,et al.  Characterization of the Human Forssman Synthetase Gene , 1999, The Journal of Biological Chemistry.

[2]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[3]  N. Shaper,et al.  Murine alpha 1,3-galactosyltransferase. A single gene locus specifies four isoforms of the enzyme by alternative splicing. , 1992, The Journal of biological chemistry.

[4]  M. Palcic,et al.  Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli. , 2001, European journal of biochemistry.

[5]  G J Barton,et al.  ALSCRIPT: a tool to format multiple sequence alignments. , 1993, Protein engineering.

[6]  M. Sippl,et al.  Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain. , 1999, Glycobiology.

[7]  J. Thornton,et al.  PROMOTIF—A program to identify and analyze structural motifs in proteins , 1996, Protein science : a publication of the Protein Society.

[8]  J. Baenziger,et al.  Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  R. Cummings,et al.  Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA. , 1990, The Journal of biological chemistry.

[10]  D. Joziasse.  Mammalian glycosyltransferases: genomic organization and protein structure. , 1992, Glycobiology.

[11]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[12]  W A Hendrickson,et al.  Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three‐dimensional structure. , 1990, The EMBO journal.

[13]  Christian Cambillau,et al.  Crystal structures of the bovine β4galactosyltransferase catalytic domain and its complex with uridine diphosphogalactose , 1999, The EMBO journal.

[14]  Thomas C. Terwilliger,et al.  Automated MAD and MIR structure solution , 1999, Acta crystallographica. Section D, Biological crystallography.

[15]  M. Palcic,et al.  Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry. , 2000, Journal of mass spectrometry : JMS.

[16]  A. V. van Tunen,et al.  Bovine alpha 1----3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA. , 1989, The Journal of biological chemistry.

[17]  Sang J. Chung,et al.  Selective inhibition of β-1,4- and α-1,3-galactosyltransferases: donor sugar-nucleotide based approach , 1999 .

[18]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[19]  E. Jabs,et al.  Characterization of an alpha 1----3-galactosyltransferase homologue on human chromosome 12 that is organized as a processed pseudogene. , 1991, The Journal of biological chemistry.

[20]  C Sander,et al.  Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme. , 1995, The EMBO journal.

[21]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[22]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[23]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[24]  S. Shohet,et al.  Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 1----3Gal epitope in primates. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. Munro,et al.  Activity of the yeast MNN1 alpha-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[26]  S. Hakomori,et al.  Sugar-nucleotide donor specificity of histo-blood group A and B transferases is based on amino acid substitutions. , 1990, The Journal of biological chemistry.

[27]  D. H. van den Eijnden,et al.  Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus. , 1985, The Journal of biological chemistry.

[28]  Linda,et al.  Frameshift and Nonsense Mutations in a Human Genomic Sequence Homologous to a Murine UDP-Gal:&D-Gal( 1,4)-D-GlcNAc CY( 1,3)-Galactosyltransferase cDNA* , 2001 .

[29]  S. Kim,et al.  Structure-based assignment of the biochemical function of a hypothetical protein: a test case of structural genomics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Sander,et al.  Protein structure comparison by alignment of distance matrices. , 1993, Journal of molecular biology.

[31]  S. Shohet,et al.  Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. , 1988, The Journal of biological chemistry.

[32]  D. Cooper.  Xenoantigens and xenoantibodies , 1998, Xenotransplantation.

[33]  P. Freemont,et al.  Crystal structure of the DNA modifying enzyme beta‐glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. , 1994, The EMBO journal.

[34]  C. Sander,et al.  Errors in protein structures , 1996, Nature.

[35]  Henrik Clausen,et al.  Molecular genetic basis of the histo-blood group ABO system , 1990, Nature.

[36]  R. Cummings,et al.  Expression Cloning of a New Member of the ABO Blood Group Glycosyltransferases, iGb3 Synthase, That Directs the Synthesis of Isoglobo-glycosphingolipids* , 2000, The Journal of Biological Chemistry.

[37]  A. Imberty,et al.  T4 phage beta-glucosyltransferase: substrate binding and proposed catalytic mechanism. , 1999, Journal of molecular biology.

[38]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[39]  S. Withers,et al.  Glycosyl Fluorides Can Function as Substrates for Nucleotide Phosphosugar-dependent Glycosyltransferases* , 1999, The Journal of Biological Chemistry.

[40]  R. Cummings,et al.  Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Rini,et al.  X‐ray crystal structure of rabbit N‐acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily , 2000, The EMBO journal.

[42]  J. Li,et al.  Alpha-Gal oligosaccharides: chemistry and potential biomedical application. , 1999, Current medicinal chemistry.

[43]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[44]  D. Joziasse,et al.  Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection. , 1999, Biochimica et biophysica acta.

[45]  S. Hakomori.  Antigen structure and genetic basis of histo-blood groups A, B and O: their changes associated with human cancer. , 1999, Biochimica et biophysica acta.

[46]  B. Rost,et al.  Combining evolutionary information and neural networks to predict protein secondary structure , 1994, Proteins.

[47]  U. Galili,et al.  Defining the minimal size of catalytically active primate alpha 1,3 galactosyltransferase: structure-function studies on the recombinant truncated enzyme. , 1994, Glycobiology.

[48]  S. Hakomori,et al.  Genomic organization of human histo-blood group ABO genes. , 1995, Glycobiology.

[49]  G. Davies,et al.  Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms. , 1999, Biochemistry.