Pig cells that lack the gene for α1-3 galactosyltransferase express low levels of the gal antigen

Sharma, Ajay; Naziruddin, Bashoo; Cui, Cunqi; Martin, Michael; Xu, Hui; Wan, Hua‐Ping; Lei, Ying; Harrison, C.; Yin, Jessie; Okabe, Jeannine; Mathews, Christine; Stark, Aileen; Adams, Connie S.; Houtz, Jeffrey; Wiseman, Barry; Byrne, Guerard W.; Logan, John S.

doi:10.1097/01.tp.0000053615.98201.77

Cited by 96 publications

(90 citation statements)

References 39 publications

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“…We attribute this Gal␣(1,3)Gal expression to an additional transferase, iGb3S. Recently, Gal␣(1,3)Gal expression was similarly reported on GGTA1 Ϫ/Ϫ pig cell lines (15) and our mAbs and human serum bind tissues from GGTA1 Ϫ/Ϫ pigs (M. S. Sandrin and A. J. d'Apice, unpublished data). We have shown that mRNA for iGb3S is expressed in pig tissues and a fucosylated form of iGb3 has been described in hog mucosa (29), suggesting that iGb3S may also be responsible for Gal␣(1,3)Gal expression in GGTA1 Ϫ/Ϫ pigs as it is in mice.…”

Section: Previous Studies Of Ggta1mentioning

confidence: 87%

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The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

Milland

Christiansen

Lazarus

et al. 2006

The Journal of Immunology

104

105

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The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes α1,3galactosyltransferase (α1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galα(1,3)Gal carbohydrate epitope was not observed as Galα(1,3)Gal staining was still present in tissues from GGTA1−/− animals. This shows that, contrary to previous dogma, α1,3GT is not the only enzyme able to synthesize Galα(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 Galα(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galα(1,3)Gal epitope. Galα(1,3)Gal synthesized by iGb3S, in contrast to α1,3GT, was resistant to down-regulation by competition with α1,2fucosyltransferase. Moreover, Galα(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 −/− mice. Galα(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.

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Section: Previous Studies Of Ggta1mentioning

confidence: 87%

“…However, several years ago, we produced anti-Gal␣ (1,3)Gal mAbs that, unlike the IB 4 lectin, stained a wide range of tissues in GGTA1 Ϫ/Ϫ mice. Recently, Sharma et al (15) showed that cells from GGTA1 Ϫ/Ϫ pig lines also express low levels of Gal␣(1,3)Gal when stained with anti-Gal␣(1,3)Gal mAbs.…”

mentioning

confidence: 99%

The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

Milland

Christiansen

Lazarus

et al. 2006

The Journal of Immunology

104

105

View full text Add to dashboard Cite

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“…40). Importantly, while the use of animals deficient in the ␣1,3-galactosyltransferase may appear to be the most attractive option for animal to human xenotransplantation, studies have suggested that some tissues, including the vascular endothelium of deficient animals, may in fact still express minor quantities of Gal␣1-3Gal epitopes (41,42). The most likely explanation for this has been identified as expression of the isogloboside glycolipid (Gal␣1-3Gal␤1-4Glc␤1-Cer) (42), which is controlled by another homologous glycosyltransferase of the CAZy GT6 family, the iGb3 synthase (UDP-Gal:lactosylceramide ␣1,3-galactosyltransferase) (43).…”

Section: Discussionmentioning

confidence: 99%

Identification of a GH110 Subfamily of α1,3-Galactosidases

Liu

Yuan

Bennett

et al. 2008

Journal of Biological Chemistry

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In search of ␣-galactosidases with improved kinetic properties for removal of the immunodominant ␣1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of ␣-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454 -464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal␣1-3(Fuc␣1-2)Gal, whereas linear oligosaccharides terminated by ␣1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal␣1-3Gal␤1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific ␣1,3-galactosidases that act equally well on both branched blood group B and linear ␣1,3Gal structures. We determined by one-dimensional 1 H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known ␣-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant ␣3Gal xenotransplantation epitope.

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“…Although no doubly targeted cell clones were obtained, a single clone with a missense mutation in the nontargeted allele was isolated. Sharma et al (18), using a similar antibody and complement selection with heterozygously targeted cells, isolated 11 resistant cell clones from a starting population totaling 2 ϫ 10 7 cells. Southern blot analysis of two of these cell clones indicated loss of the nontargeted allele although the mechanism of loss was not further investigated and no pigs were produced by using the antibody resistant lines.…”

Section: Discussionmentioning

confidence: 99%

Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations

Kolber-Simonds

Lai

Watt

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

422

338

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Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the ␣-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the genetargeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover͞gene conversion events, respectively. Aside from loss of Gal␣-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.A ntibodies against galactosyl-␣-1,3-galactose (Gal␣-1,3-Gal) residues on cell surface glycoproteins of pig cells mediate hyperacute rejection of porcine organs in primate model recipients and are the most immediate barrier to successful clinical xenotransplantation (1, 2). High levels of preformed ''natural'' antibodies against the Gal␣-1,3-Gal epitope are found in humans and old world primates, following evolutionary loss of the corresponding galactosyltransferase activity (encoded by GGTA1) (3). The presence of these antibodies, along with the high density of Gal␣-1,3-Gal residues on most pig cells (4), suggests that elimination of GGTA1 function would provide a practical means of overcoming both hyperacute rejection and subsequent acute or chronic tissue damage associated with antibody binding to this epitope.The lack of GGTA1 function in humans and old world primates, along with the viability of GGTA1 knockout mice produced with embryonic stem cell technology (5, 6), suggested that a knockout strategy might be biologically feasible in pigs. The cloning of sheep (7) and subsequently pigs (8-10) by nuclear transfer with somatic cells has made attempts to knockout the GGTA1 locus in pigs technically feasible.We have previously reported the generation of GGTA1 heterozygous inbred miniature swine using nuclear transfer with gene-targeted fibroblasts (11). Starting with heterozygous fibroblasts from such animals, we now report the isolation of GGTA1 null cells with spontaneous loss of the WT allele. The rate of loss of heterozygosity (LOH) was several orders of magnitude greater than typically expected, an observation that may be related to the inbred background of the heterozygous animals. LOH resulted in some cases from deletion of the WT allele and in others from either somatic crossing over or gene conversion. Similarly high rates of somatic recombi...

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Pig cells that lack the gene for α1-3 galactosyltransferase express low levels of the gal antigen

Abstract: Fetal-pig fibroblasts homozygous for the knockout of the alpha1-3 galactosyltransferase gene appear to express low but detectable levels of the gal antigen.

Cited by 96 publications

References 39 publications

The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

The Molecular Basis for Galα(1,3)Gal Expression in Animals with a Deletion of the α1,3Galactosyltransferase Gene

Identification of a GH110 Subfamily of α1,3-Galactosidases

Production of α-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations

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