Adenovirus－mediated expression of pig α（1，3）galactosyltransferase reconstructs Gal α（1,3） Gal epitope on the surface of human tumor cells

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.     

Efficient generation of alpha(1,3) galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer

Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the (1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/– somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.     

The expression of beta-1,3 galactosyltransferase and beta-1,4 galactosyltransferase enzymatic activities in the mammary gland of the tammar wallaby (Macropus eugenii) during early lactation

The regulation of beta-1,3 galactosyltransferase (3betaGalT) and beta-1,4 galactosyltransferase enzymatic (4betaGalT) activities in the mammary gland of the tammar wallaby (Macropus eugenii) have been characterised. These two beta-galactosyltransferases are active at different times during the lactation cycle and play a central role in regulating the carbohydrate composition in tammar milk, which changes progressively throughout lactation to assist the physiological development of the altrical young. The 4betaGalT activity was present at parturition and increased 3-fold by day 10 of lactation (d10L), whereas 3betaGalT activity was barely detectable at day d5L and then increased 6-fold by d10L. This increase in activity of both enzymes was sucking dependent. While 3betaGalT activity was not observed in the mammary gland prior to d7L, this activity was found in mammary explants from late pregnant tammar cultured with insulin, hydrocortisone and prolactin (IFP) and was further stimulated by the addition of tri-iodothyronine (T) and 17beta-oestradiol (E). The activity of 4betaGalT in these explants was stimulated maximally with IFP. These data suggest the temporal activity of both 3betaGalT and 4betaGalT is most likely regulated by both endocrine stimuli and factors intrinsic to the mammary gland.     

Chromosomal localization of the gene for a human galactosyltransferase (GT-1)

The gene for human galactosyltransferase (EC 2.4.1.22) has been localized to the short arm of chromosome 9 by in situ hybridization to human metaphase chromosomes of a 985 bp cDNA probe for the gene.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells

The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.     

Isolation of a novel human alpha (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group alpha (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities.

Biochemical and genetic evidence indicates that the human genome may encode four or more distinct GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha(1,3)fucosyltransferase) activities. Genes encoding two of these activities have been previously isolated. These correspond to an alpha(1,3/1,4)fucosyltransferase thought to represent the human Lewis blood group locus and an alpha(1,3)fucosyltransferase expressed in the myeloid lineage. We report here the molecular cloning and expression of a third human alpha(1,3)fucosyltransferase gene, homologous to but distinct from the two previously reported human fucosyltransferase genes. When expressed in transfected mammalian cells, this gene determines expression of a fucosyltransferase capable of using N-acetyllactosamine to form the Lewis x epitope, and alpha(2,3)sialyl-N-acetyllactosamine to construct the sialyl Lewis x moiety. This enzyme shares 91% amino acid sequence identity with the human Lewis blood group alpha(1,3/1,4)fucosyltransferase, yet exhibits only trace amounts of alpha(1,4)fucosyltransferase activity. Polymerase chain reaction analyses were used to demonstrate that the gene is syntenic to the Lewis locus on chromosome 19. These analyses also excluded the possibility that this DNA segment represents an allele of the Lewis locus that encodes alpha(1,3)fucosyltransferase but not alpha(1,4)fucosyltransferase activity. These results are consistent with the hypothesis that this gene encodes the human "plasma type" alpha(1,3)fucosyltransferase, and suggest a molecular basis for a family of human alpha(1,3)fucosyltransferase genes.     

An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.     

Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides.

alpha1,3galactosyltransferase (alpha1,3GalT) catalyzes the synthesis of a range of glycoconjugates containing the Galalpha1,3Gal epitope which is recognized by the naturally occurring human antibody, anti-Gal. This enzyme may be a useful synthetic tool to produce a range of compounds to further investigate the binding site of anti-Gal and other proteins with a Galalpha1,3Gal binding site. Thus, the enzyme has been probed with a series of type 2 disaccharide-C8(Galbeta1-4GlcNAc-C8) analogs. The enzyme tolerated acceptors with modifications at C2 and C3 of the N-acetylglucosamine residue, producing a family of compounds with a nonreducing alpha1,3 linked galactose. Compounds that did not serve as acceptors were evaluated as inhibitors. Interestingly, the type 1 disaccharide-C8, Galbeta1-3GlcNAc-C8, was a good inhibitor of the enzyme (Ki = 270 microM vs. Km = 190 microM for Galbeta1-4GlcNAc-C8). A potential photoprobe, based on a modified type 2 disaccharide (octyl 3-amino-3-deoxy-3-N-(2-diazo-3, 3, 3-trifluoropropionyl-beta-D-galactopyranosyl-(1, 4)-2-acetamindo-2-deoxy-beta-D-glycopyranoside, (DTFP-LacNAc-C8)), was evaluated as an inhibitor of alpha1,3GalT. alpha1,3GalT bound DTFP-LacNAc-C8 with an affinity (Ki = 300 microM) similar to that displayed by the enzyme for LacNAc-C8. Additional studies were done to determine the enzyme's ability to transfer a range of sugars from UDP-sugar donors. The results of these experiments demonstrated that alpha1,3GalT has a strict specificity for UDP-Gal. Finally, inactivation studies with various amino acid modifiers were done to obtain information on the importance of different types of amino acids for alpha1,3GalT activity.     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.     

Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.     

The molecular basis for the B(A) allele: an amino acid alteration in the human histoblood group B alpha-(1,3)-galactosyltransferase increases its intrinsic alpha-(1,3)-N-acetylgalactosaminyltransferase activity.

The formation of the subgroup B(A) phenotype is thought to be due to an overlapping specificity of the human blood group A and B transferases. A new molecular basis for the B(A) allele, resulting from the C(700) to G substitution which predicts the alteration of Pro(234) to Ala, just ahead of the second of the four amino acid residues which differentiates the specificities of the A and B transferases, is reported here. Compared to normal group B sera, a relatively lower B-transferase activity was demonstrated in the B(A) serum, which correlated well with the observation of a smaller amount of B antigen on the B(A) red cells. Also a much higher A-transferase activity was demonstrated in the B(A) serum in contrast to the minute amount of A-transferase activity found in normal group B sera. The formation of the B(A) phenotype in this report is most likely due to the shifting of the specificity of the B transferase rather than an enhanced B-transferase activity which was previously presumed to be responsible for the formation of this phenotype. The Pro(234) to Ala alteration is suggested to be responsible for the shifting of the specificity with a subsequent increase in A- but a decrease in B-transferase activity. This new B(A) allele shows that not only the four critical residues but also the neighboring areas may influence the specificity of the A and B transferases.