Molecular Cloning of Endo-β-galactosidase C and Its Application in Removing α-Galactosyl Xenoantigen from Blood Vessels in the Pig Kidney

Ogawa, Hideoki; Muramatsu, Hisako; Kobayashi, Takaaki; Morozumi, Kunio; Yokoyama, Itsuo; Kurosawa, Nobuyuki; Nakao, Akimasa; Muramatsu, Takashi

doi:10.1074/jbc.m001888200

Cited by 51 publications

(50 citation statements)

References 33 publications

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“…The inverting nature of family GH110 enzymes excludes such undesired reactions. Considerable efforts have been applied to testing the use of an endo-␤-galactosidase with strict specificity for the Gal␣1-3Gal␤1-4GlcNAc structure (35,(37)(38)(39). The action of this enzyme results in exposure of ␤GlcNAc residues rather that the Gal␤1-4GlcNAc structures exposed with exo-␣-galactosidase strategies, and this may result in other immune problems.…”

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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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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“…It is noteworthy that E-ABase does not share significant sequence homology with other endo-␤-galactosidases cloned from Flavobacterium keratolyticus (DDBJ/GenBank TM /EBI accession number AF083896) (31) and C. perfringens (accession numbers AB038772 and AB059351) (25,32). The fact that the primary sequence of E-ABase does not contain an EXDX(X)E motif as found in other endo-␤-galactosidases (25, 31, 32) indicates that E-ABase may have evolved along a separate evolutionary line.…”

Section: Assignment Of E-abase To a New Glycoside Hydrolase Family Gh98mentioning

confidence: 99%

A Clostridial Endo-β-galactosidase That Cleaves Both Blood Group A and B Glycotopes

Anderson

Ashida

Maskos

et al. 2005

Journal of Biological Chemistry

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We have isolated an endo-␤-galactosidase designated E-ABase from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide (A-Tri; GalNAc␣133(Fuc␣132)Gal) and B trisaccharide (B-Tri; Gal␣133(Fuc␣132)Gal) from glycoconjugates containing blood group A and B glycotopes, respectively. We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism. This gene was found to be identical to the CPE0329 gene of C. perfringens strain 13, whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H. human ovarian cyst glycoprotein were established by NMR spectroscopy. The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A-and B-containing glycoconjugates as well as for identifying other glycosidases belonging to the new GH98 family.

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“…It contains two EndoGalC expression units, in which EndoGalC expression is regulated by two different promoters, CAG (comprising cytomegalovirus enhancer and chicken β-actin promoter; [23]) and mouse Pol II promoter [25]. The CAGEndoGalC expression unit isolated from pCAG/GT+Endo [12] was combined with the Pol II-EndoGalC expression unit in a head-tohead fashion (Fig. 1A).…”

Section: Vector Constructionmentioning

confidence: 99%

“…This enzyme is derived from Clostridium perfringens and can digest the α-Gal epitope by cleaving the α-galactosidic linkage [11]. EndoGalC is thus considered an effective means of enzymatic removal of the α-Gal epitope from the surface of animal cells such as porcine cells [12][13][14]. However, no attempt to produce genetically engineered pigs expressing EndoGalC systemically has yet been made.…”

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confidence: 99%

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Production of Genetically Modified Porcine Blastocysts by Somatic Cell Nuclear Transfer: Preliminary Results Toward Production of Xenograft-Competent Miniature Pigs

Himaki

Watanabe

Chi

et al. 2010

Journal of Reproduction and Development

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Abstract. Galα1-3Gal (α-Gal epitope) is the major xenoantigenic epitope responsible for hyperacute rejection upon pigto-human xenotransplantation. Endo-β-galactosidase C (EndoGalC) from Clostridium perfringens can digest the α-Gal epitope. In this study, gene-engineered primary cultured porcine embryonic fibroblasts (PEF) expressing EndoGalC were obtained and subjected to somatic cell nuclear transfer (SCNT) to test whether xenograft-competent pigs can be created. The EndoGalC-expressing PEF clones exhibited highly reduced expression of α-Gal epitope, as revealed by cytochemical staining with BS-I-B4 isolectin, a lectin that specifically binds to α-Gal epitope, and FACS analysis. The pattern of low level of α-Gal epitope expression continued for at least 6 months (more than 10 generations) after isolation. SCNT of nuclei from these cells resulted in the generation of blastocysts that displayed nearly complete loss of α-Gal epitope from their cell surface. This is the first study to demonstrate that SCNT using EndoGalC-expressing PEFs as donors would be useful for production of genetically modified cloned pigs suitable for xenotransplantation. Key words: Endo-β-galactosidase C, Gene-modified, Miniature pig, Somatic cell nuclear transfer, Xenotransplantation (J. Reprod. Dev. 56: [630][631][632][633][634][635][636][637][638] 2010) ig-to-human xenotransplantation leads to an immediate rejection process, termed hyperacute rejection (HAR), caused by the binding of natural antibodies present in humans to the carbohydrate epitope gal α-1,3-gal (α-Gal epitope) that is present in pigs but not in humans [1][2][3]. This epitope is synthesized mainly by α-1,3-galactosyltransferase (α-1,3-GalT-1), and removal of the epitope is considered a prerequisite for xenotransplantation [4].Several approaches have been proposed to eliminate the α-Gal epitope from pigs. Removal of the α-1,3-GalT-1 gene by gene targeting is a straightforward method. In fact, in pigs, successful gene targeting has been achieved by a somatic cell nuclear transfer (SCNT) technique using targeted fibroblast clones [5][6][7][8][9][10]. Another approach is employment of endo-β-galactosidase C (EndoGalC). This enzyme is derived from Clostridium perfringens and can digest the α-Gal epitope by cleaving the α-galactosidic linkage [11]. EndoGalC is thus considered an effective means of enzymatic removal of the α-Gal epitope from the surface of animal cells such as porcine cells [12][13][14]. However, no attempt to produce genetically engineered pigs expressing EndoGalC systemically has yet been made.Several approaches have been considered for the production of pigs overexpressing the EndoGalC gene. [19][20][21][22]. Of these approaches, the SCNT-mediated transgenesis approach appears to be most promising, since donor cells highly expressing a transgene can easily be selected prior to SCNT. In this case, the most important point requiring consideration is how EndoGalC is ubiquitously expressed through each organ of the SCNT pig. We therefore employed two types of...

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Molecular Cloning of Endo-β-galactosidase C and Its Application in Removing α-Galactosyl Xenoantigen from Blood Vessels in the Pig Kidney

Cited by 51 publications

References 33 publications

Identification of a GH110 Subfamily of α1,3-Galactosidases

Identification of a GH110 Subfamily of α1,3-Galactosidases

A Clostridial Endo-β-galactosidase That Cleaves Both Blood Group A and B Glycotopes

Production of Genetically Modified Porcine Blastocysts by Somatic Cell Nuclear Transfer: Preliminary Results Toward Production of Xenograft-Competent Miniature Pigs

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