Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells

Xenotransplantation from pig to human being is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal were capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.     

Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells

Xenotransplantation from pigs to human beings is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (a Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and the porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal was capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and the human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.     

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

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.     

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.     

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.     

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.     

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.     

Interleukin-1beta induces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene

We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.     

Carbohydrate residues downstream of the terminal Galalpha(1,3)Gal epitope modulate the specificity of xenoreactive antibodies

Carbohydrates are involved in many immunological responses including the rejection of incompatible blood, tissues and organs. Carbohydrate antigens with Galalpha(1,3)Gal epitopes are recognized by natural antibodies in humans and pose a major barrier for pig-to-human xenotransplantation. Genetically modified pigs have been established that have no functional alpha1,3-galactosyltransferase (alpha1,3GT), which transfers alphaGal to N-acetyllactosamine (LacNAc) type oligosaccharides. However, a low level of Galalpha(1,3)Gal is still expressed in alpha1,3GT knockout animals in the form of a lipid, isoglobotrihexosylceramide (iGb3), which is produced by iGb3 synthase on lactose (Lac) type core structures. Here, we define the reactivity of a series of monoclonal antibodies (mAb) generated in alpha1,3GT-/- mice immunized with rabbit red blood cells (RbRBC), as a rich source of lipid-linked antigens. Interestingly, one mAb (15.101) binds weakly to synthetic and cell surface-expressed Galalpha(1,3)Gal on LacNAc, but strongly to versions of the antigen on Lac cores, including iGb3. Three-dimensional models suggest that the terminal alpha-linked Gal binds tightly into the antibody-binding cavity. Furthermore, antibody interactions were predicted with the second and third monosaccharide units. Collectively, our findings suggest that although the terminal carbohydrate residues confer most of the binding affinity, the fine specificity is determined by subsequent residues in the oligosaccharide.     

Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection

The transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galalphal,3Gal, which is synthesized by Galbeta1,4GlcNAc alpha1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of alpha1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking alpha1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of alpha1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses.     

Secretion of a1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera

Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Galβ1→4GLcNAc (Gal to Gal) a1,3 galactosyltransferase (a1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the a1,3GT activity. A recombinant and soluble form of the murine a1,3GT was produced in H293 cells (H293-a1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-a-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, a1,3GT and the time of incubation. The assay was also useful in monitoring a1,3GT activity during enzyme enrichment procedures. Using this assay, we found that a1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the a1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon. This revised version was published online in November 2006 with corrections to the Cover Date.     

Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.     

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.     

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)