Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

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

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.     

Alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Gal beta 1----4GlcNAc alpha 1----3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active alpha 1----3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant alpha 1----3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine-Sepharose. The recombinant alpha 1----3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Gal beta 1----4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus alpha 1----3-galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927-12934]. In pilot studies, the recombinant enzyme was examined for the ability to synthesize alpha 1----3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H-NMR, methylation analysis, HPLC, and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Gal alpha 1----3Gal beta 1----4-R. Our results demonstrate that catalysis by recombinant alpha 1----3-galactosyltransferase can be used to obtain preparative quantities of various alpha 1----3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.     

Isolation of the feline alpha1,3-galactosyltransferase gene, expression in transfected human cells and its phylogenetic analysis

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.     

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

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Regulation of the expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in kidney

Previous studies (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373; Galili, U., Shohet, S. B., Korbrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762) have established that there is a unique evolutionary distribution of glycoconjugates carrying the Gal alpha 1-3Gal beta 1-4GlcNAc epitope. These glycoconjugates are expressed by cells from New World monkeys and non-primate mammals, but not by cells from humans, Old World monkeys, or apes. The lack of expression of this epitope in the latter species appears to result from the suppression of gene expression for the enzyme UDP-galactose:nLc4Cer alpha 1-3-galactosyltransferase (alpha 1-3GalT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Although many non-primate species are known to express this carbohydrate epitope, the nature (i.e. glycoprotein or glycosphingolipid) of the glycoconjugate carrying this epitope is only known for a few tissues in a few animal species. Furthermore, it is not known whether all animal species express this epitope in the same tissues. We have investigated these questions by analyzing the glycosphingolipids in kidney from several non-primate animal species. Immunostained thin layer chromatograms of glycosphingolipids from sheep, pig, rabbit, cow, and rat kidney with the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipid-specific monoclonal antibody, Gal-13, demonstrated that kidney from all of these species except rat contained Gal alpha 1-3Gal beta 1-4GlcNAc neutral glycosphingolipids. A lack of expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in rat may be due to the lack of expression of the enzyme (alpha 1-3GalT) which catalyzes the formation of the Gal alpha 1-3Gal nonreducing terminal sequence of these compounds or to the lack of expression of glycosyltransferases which are necessary for the synthesis of the neolacto core structure of these compounds. These possibilities were evaluated in two ways. First, the three enzymes (UDP-N-acetylglucosamine:LacCer beta 1-3-N-acetyl-glucosaminyltransferase, UDP-galactose:Lc3Cer beta 1-4-galactosyltransferase, and alpha 1-3GalT) involved in the synthesis of the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids were assayed using an enzyme-linked immunosorbent assay-based assay system and carbohydrate sequence-specific monoclonal antibodies. Second, TLC immunostaining was done to determine if the glycosphingolipid precursors (i.e. Lc3Cer and nLc4Cer) are expressed in rat kidney. Interestingly, rat kidney had a relatively high level of alpha 1-3GalT activity compared with the other animals tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.     

Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.     

Primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase determined by mass spectrometry sequence analysis and molecular cloning. Evidence for a protein motif in the sialyltransferase gene family.

The Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase forms the NeuAc alpha 2,3Gal beta 1,3(4)GlcNAc sequences found in terminal carbohydrate groups of glycoproteins and glycolipids. High energy collision-induced dissociation analysis of tryptic peptides from only 300 pmol of the purified Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase provided 25% of the total amino acid sequence and led to the successful cloning of this enzyme. The peptide sequence information was used to design short degenerate primers for use in the polymerase chain reaction. A long specific cDNA fragment was amplified which was used to isolate a clone from a rat liver cDNA library. The cloned cDNA encodes a 374-amino acid protein containing an amino-terminal signal-anchor sequence characteristic of all cloned glycosyltransferases and produced sialyltransferase activity when transiently expressed in COS-1 cells. When compared with two other cloned sialyltransferases, the primary structure of Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase revealed a homologous region in all three enzymes consisting of a stretch of 55 amino acids located in their catalytic domains. This feature together with lack of homology in the remaining 85% of the sequence of the three sialyltransferases defines a pattern of sequence homology not found in cloned cDNAs of other glycosyltransferase families.     

Activation of two new alpha(1,3)fucosyltransferase activities in Chinese hamster ovary cells by 5-azacytidine.

Several mammalian alpha(1,3)fucosyltransferases (alpha[1,3]Fuc-T) that synthesize carbohydrates containing alpha(1,3)fucosylated lactosamine units have been identified. Although Chinese hamster ovary (CHO) cells do not express alpha(1,3)Fuc-T activity, the rare mutants LEC11 and LEC12, isolated after mutagenesis or DNA transfection, each express an alpha(1,3)Fuc-T that may be distinguished by several criteria. Two new CHO mutants possessing alpha(1,3)Fuc-T activity (LEC29 and LEC30) have now been isolated after treatment of a CHO cell population with 5-azacytidine (5-AzaC), ethylnitrosourea (ENU), or 5-AzaC followed by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Like LEC12, both mutants possess an N-ethylmaleimide-resistant alpha(1,3)Fuc-T activity that can utilize a variety of acceptors and both express the Lewis X (Lex) determinant (Gal beta[1,4](Fuc alpha[1,3])GlcNAc beta 1)) but not the sialyl alpha(2,3)Lex determinant on cell-surface carbohydrates. However, LEC29 and LEC30 may be distinguished from LEC11 and LEC12, as well as from each other, on the basis of their unique patterns of lectin resistance and their abilities to bind the VIM-2 monoclonal antibody that recognizes carbohydrates terminating in NeuNAc alpha(2,3)Gal beta(1,4)GlcNAc beta(1,3)Gal beta(1,4)(Fuc alpha[1,3])GlcNAc beta and also by the different in vitro substrate specificities and kinetic properties of their respective alpha(1,3)Fuc-T activities. The combined data provide good evidence that the LEC29 and LEC30 alpha(1,3)Fuc-Ts are novel transferases encoded by distinct gene products.     

Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.     

Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase

The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.     

Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay

The study of the expression of Gal alpha 1----3Gal beta 1----4GlcNAc residues on mammalian glycoconjugates is of particular interest since as many as 1% of circulating IgG antibodies in man (the natural anti-Gal antibody) interact specifically with this carbohydrate residue. In recent studies, we have found that Gal alpha 1----3Gal beta 1----4GlcNAc residues are abundant on red cells and nucleated cells of nonprimate mammals, prosimians, and New World monkeys, but their expression is diminished in Old World monkeys, apes, and humans. In the present work, we have analyzed the expression of these residues on secreted mammalian glycoproteins. For this purpose, we have developed a radioimmunoassay (RIA) which enables the quantification of Gal alpha 1----3Gal beta 1----4GlcNAc residues on the secreted glycoproteins. Purified biotinylated anti-Gal was used as the antibody in the RIA, and bovine thyroglobulin enriched for Gal alpha 1----3Gal beta 1----4GlcNAc residues served as a solid-phase antigen. In this study, it is reported for the first time that the evolutionary pattern of Gal alpha 1----3Gal beta 1----4GlcNAc residue distribution in in vivo secreted glycoproteins is similar to that observed in membranes of cell lines and of red cells. Thyroglobulin, fibrinogen, or IgG molecules from nonprimate mammals and from New World monkeys express varying amounts of Gal alpha 1----3Gal beta 1----4GlcNAc residues ranging between 0.01 and 11 residues per molecule, whereas no such residues are present on any of these glycoproteins of human or Old World monkey origin.(ABSTRACT TRUNCATED AT 250 WORDS)