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Why humans and Catarrhini lack the Galα1-3Gal epitope, related to xenograft rejection?  [PDF]
Anna Suchanowska,Marcin Czerwiński
Post?py Higieny i Medycyny Do?wiadczalnej , 2009,
Abstract: The Galα1-3Gal epitope (Galβ1-3Galα1-4GlcNAc-R) is an oligosaccharide determinant present on the cell surface of most mammalian species with the exception of the higher primates, including Old World monkeys, apes, and humans. The synthesis of Galα1-3Gal epitope is catalyzed by α1,3-galactosyltransferase (α1,3GT). Inactivation of the α1,3GT gene in humans and the production of natural anti-Gal 1-3Gal antibodies against the Galα1-3Gal epitope has resulted in the formation of a unique immunological barrier that prevents the transplantation of tissues and organs from Galα1-3Gal-positive animals to humans. The gene encoding α1,3-galactosyltransferase in higher primates is inactive due to point mutations and deletions leading to a change of reading frame. The human transcript of this gene consists of several splicing variants, most of which does not contain an exon encoding the catalytic domain. Thus no active protein is produced.
Proteomic Analysis of Tissue from α1,3-galactosyltransferase Knockout Mice Reveals That a Wide Variety of Proteins and Protein Fragments Change Expression Level  [PDF]
Louise Thorlacius-Ussing, Maja Ludvigsen, Svend Kirkeby, Henrik Vorum, Bent Honoré
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0080600
Abstract: A barrier in a pig-to-man xenotransplantation is that the Galα1-3Galβ1-4GlcNAc-R carbohydrate (α-Gal epitope) expressed on pig endothelial cells reacts with naturally occurring antibodies in the recipient’s blood leading to rejection. Deletion of the α1,3-galactosyltransferase gene prevents the synthesis of the α-Gal epitope. Therefore, knockout models of the α1,3-galactosyltransferase gene are widely used to study xenotransplantation. We have performed proteomic studies on liver and pancreas tissues from wild type and α1,3-galactosyltransferase gene knockout mice. The tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis and liquid chromatography - tandem mass spectrometry. The analyses revealed that a wide variety of proteins and protein fragments are differentially expressed suggesting that knockout of the α1,3-galactosyltransferase gene affects the expression of several other genes.
Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope
Anne M Ruffing, Rachel R Chen
Microbial Cell Factories , 2010, DOI: 10.1186/1475-2859-9-1
Abstract: Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac.The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.α-Gal epitopes are oligosaccharides containing terminal Gal-α1,3-Gal residues. In nature, three main α-Gal epitopes are produced: two trisaccharides (Gal-α1,3-Gal-β1,4-GlcNAc and Gal-α1,3-Lac) and a pentasaccharide (Gal-α1,3-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc). These epitopes are components of glycolipids and glycoproteins displayed on the cell surface of non-primate mammals and New World monkeys via expression of an α1,3-galactosyltransferase (α1,3-GalT). The α1,3-GalT was inactivated in ancestral Old World primates approximately 20-28 million years ago, resulting in the absence of α-Gal epitopes in humans, apes, and Old World monkeys today [1,2]. These evolutionary descendents of Old World
ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines
Paula A Videira, Manuela Correia, Nadia Malagolini, Hélio J Crespo, Dário Ligeiro, Fernando M Calais, Helder Trindade, Fabio Dall'Olio
BMC Cancer , 2009, DOI: 10.1186/1471-2407-9-357
Abstract: Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: ST3Gal.I, ST3Gal.II and ST3Gal.IV mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines.In nonmuscle-invasive bladder cancers, ST3Gal.I mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, ST3Gal.I mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed ST3Gal.I mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the ST3Gal.I mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two ST3Gal.I transcript variants were also equally expressed, independently of cell phenotype or malignancy.ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of ST3Gal.I seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.Bladder cancer is one of the most common cancers in humans and its incidence has been increasing during the past years [1]. 70-80% of all bladder cancers are nonmuscle-invasive and have a low mortality rate. However, despite the complete resection of the primary lesion, and the success of treatments such as the intravesical instillation with bacillus Calmette-Guérin (BCG) [2], 30-50% of patients with nonmuscle invasive bladder cancer experience recur
α2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro and Enhances Its Metastatic Potential In Vivo  [PDF]
Marta Pérez-Garay,Beatriz Arteta,Lluís Pagès,Rafael de Llorens,Carme de Bolòs,Fernando Vidal-Vanaclocha,Rosa Peracaula
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0012524
Abstract: Cell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process.
Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing
Marion Lanteri, Laurence Ollier, Valérie Giordanengo, Jean-Claude Lefebvre
Breast Cancer Research , 2005, DOI: 10.1186/bcr1034
Abstract: Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4.We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression.Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression.The enzyme α1,3galactosyltransferase (αGalT) is responsible for the synthesis of galactose-α1,3galactose-β1,4N-acetylglucosamine-R (αGal) epitopes in all mammals except Old World primates [1]. Highly expressed in nonprimate mammals, prosimians and New World monkeys, this glycosyltransferase has been mutationally inactivated during the course of evolution, starting from Old World primates [2]. We have previously shown that, in human cells, transcription of the αGalT gene is interrupted by the presence of a strong stop signal in exon 7, which leads to a chimeric mRNA comprising the first four coding exons and part of intron VII, but lacking the last two exons corresponding
The αGal Epitope of the Histo-Blood Group Antigen Family Is a Ligand for Bovine Norovirus Newbury2 Expected to Prevent Cross-Species Transmission  [PDF]
Maha Zakhour,Nathalie Ruvo?n-Clouet,Annie Charpilienne,Brigitte Langpap,Didier Poncet,Thomas Peters,Nicolai Bovin,Jacques Le Pendu
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000504
Abstract: Among Caliciviridae, the norovirus genus encompasses enteric viruses that infect humans as well as several animal species, causing gastroenteritis. Porcine strains are classified together with human strains within genogroup II, whilst bovine norovirus strains represent genogroup III. Various GI and GII human strains bind to carbohydrates of the histo-blood group family which may be shared among mammalian species. Genetic relatedness of human and animal strains as well as the presence of potentially shared ligands raises the possibility of norovirus cross-species transmission. In the present study, we identified a carbohydrate ligand for the prototype bovine norovirus strain Bo/Newbury2/76/UK (NB2). Attachment of virus-like particles (VLPs) of the NB2 strain to bovine gut tissue sections showed a complete match with the staining by reagents recognizing the Galα1,3 motif. Alpha-galactosidase treatment confirmed involvement of a terminal alpha-linked galactose. Specific binding of VLPs to the αGal epitope (Galα3Galβ4GlcNAcβ-R) was observed. The binding of Galα3GalαOMe to rNB2 VLPs was characterized at atomic resolution employing saturation transfer difference (STD) NMR experiments. Transfection of human cells with an α1,3galactosyltransferase cDNA allowed binding of NB2 VLPs, whilst inversely, attachment to porcine vascular endothelial cells was lost when the cells originated from an α1,3galactosyltransferase KO animal. The αGal epitope is expressed in all mammalian species with the exception of the Hominidaea family due to the inactivation of the α1,3galactosyltransferase gene (GGTA1). Accordingly, the NB2 carbohydrate ligand is absent from human tissues. Although expressed on porcine vascular endothelial cells, we observed that unlike in cows, it is not present on gut epithelial cells, suggesting that neither man nor pig could be infected by the NB2 bovine strain.
Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2-fucosyltransferase gene
Gong Feng,Zhang Yangpei,Jia Yanjun,Wang Yingli,Tan Yingxia,Tian Shuguang
Chinese Science Bulletin , 2005, DOI: 10.1007/BF02899642
Abstract: The polycarbohydrate structure of Galα1-3Galβ1-4GluNAc-R (known as αGal epitopes of xenoantigen), produced by α1-3-galactosyltransferase (α1,3-GT) in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosidase (AGL), a hydrolytic enzyme, can remove the terminal α-1,3-galactosyl from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGal epitopes from the porcine cells. Alpha-1,2-fucosyltransferase (HT) can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGal epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibro-blast (PFFb) in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGal xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated byAGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic cell.
Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family
Yuxin Fan,Long Yu,Qi Zhang,Ying Jiang,Fangyan Dai,Chiyuan Chen,Qiang Tu,Anding Bi,Yuefang Xu,Shouyuan Zhao
Science China Life Sciences , 1999, DOI: 10.1007/BF02882052
Abstract: By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.
Molecular Cloning, Prokaryotic Expression and Biological Activity of α-2,6 Sialyltransferase

HOU Xiao-Qiang Xian-Zhu XIA,

中国生物工程杂志 , 2009,
Abstract: 克隆载体中,经序列测定后与 GenBank 中报道的已知序列比对完全一致,再将已知目的序列插入原核表达载体pET-28α(+)中.将构建正确的原核表达载体转化表达受体菌BL21(DE3)中,IPTG诱导其进行表达并鉴定目的蛋白,对鉴定正确的目的蛋白复性后经Ni2+亲和层析柱纯化获得高纯度的α-2,6 唾液酸转移酶蛋白.然后用霍乱弧菌神经氨酸酶处理健康鸡红细胞,清除鸡红细胞表面所有类型的唾液酸寡糖链;再用表达的α-2,6 唾液酸转移酶以CMP-唾液酸作为底物在霍乱弧菌神经氨酸酶处理的鸡红细胞表面标记上SAα2,6 Gal受体.将标记有SAα2,6Gal受体的鸡红细胞应用微量血凝试验和流式细胞仪进行检测,以验证所表达蛋白的生物活性.结果表明,原核表达的α-2,6唾液酸转移酶具有较好的生物活性.
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