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.

Blanken, Willem M.; Eijnden, Dirk H. van den

doi:10.1016/s0021-9258(17)38814-2

Cited by 173 publications

(22 citation statements)

References 60 publications

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“…1). The substrate specificity of the Ul,3-galactosyltransferase has been examined in vitro, and is any unbranched galactose in a ~-linkage with either N-acetyl glucosamine or N-acetyl galactosamine [40]. Approaches to inhibit the action of this enzyme can focus on the gene or on the expression of the mRNA (Table 1, Fig.…”

Section: Gene Homologous Recombination Approaches To Xenotransplantation «13-galactosyltransferase Enzymementioning

confidence: 99%

Overcoming the Anti-Galα(1–3)Gal Reaction To Avoid Hyperacute Rejection: Molecular Genetic Approaches

Sandrin¹,

Cohney²,

Osman³

1997

Xenotransplantation

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Section: Gene Homologous Recombination Approaches To Xenotransplantation «13-galactosyltransferase Enzymementioning

confidence: 99%

Overcoming the Anti-Galα(1–3)Gal Reaction To Avoid Hyperacute Rejection: Molecular Genetic Approaches

Sandrin¹,

Cohney²,

Osman³

1997

Xenotransplantation

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“…The epitope Galα1→3Gal is formed by a reaction catalyzed by α1,3-galactosyltransferase (α1, , an enzyme encoded by the GGTA1 gene. The α1,3-GT enzyme catalyzes the galactose transfer reaction from UDP-Gal and its α1→3 glycosidic bond with glycoproteins or glycosphingolipids containing terminal Galβ1→4GlcNAc-R residues [7]. One of the strategies to remove the Galα1→3Gal epitope from the porcine cells involves combined transgenic expression of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and αgalactosidase A (rhα-Gal A) enzymes encoded by hFUT2 and hGLA genes, respectively [8].…”

Section: Introductionmentioning

confidence: 99%

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

Wiater

Samiec

Skrzyszowska

et al. 2021

IJMS

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This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA−) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA− cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA− hFUT2×hGLA cells as compared to the TSA− nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.

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“…The biogenesis of Galα1→3Gal antigenic determinants is enzymatically catalyzed by α1,3-galactosyltransferase (α1,3-GT), which is encoded by GGTA1 gene. The α1,3-GT enzyme displays the capability to biocatalyse the biochemical reactions of transferring galactose moieties from uridine 5′-diphosphogalactose (UDP-Gal) residues followed by α1→3 glycosidic binding these monosaccharide molecules to glycoproteins or glycosphingolipids containing terminal Galβ1→4GlcNAc-R residues [ 9 , 10 ]. Therefore, genetically engineering triggering the simultaneous coexpression of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) enzymes, which are encoded by h FUT2 and h GLA transgenes, appears to be a promising approach to overcome the HAR of porcine cell, tissue, and organ xenotransplants [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

Wiater

Samiec

Wartalski

et al. 2021

IJMS

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Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.

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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.

Cited by 173 publications

References 60 publications

Overcoming the Anti-Galα(1–3)Gal Reaction To Avoid Hyperacute Rejection: Molecular Genetic Approaches

Overcoming the Anti-Galα(1–3)Gal Reaction To Avoid Hyperacute Rejection: Molecular Genetic Approaches

Trichostatin A-Assisted Epigenomic Modulation Affects the Expression Profiles of Not Only Recombinant Human α1,2-Fucosyltransferase and α-Galactosidase A Enzymes But Also Galα1→3Gal Epitopes in Porcine Bi-Transgenic Adult Cutaneous Fibroblast Cells

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

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