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Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2000 Jun 23; Vol. 275 (25), pp. 19368-74. - Publication Year :
- 2000
-
Abstract
- Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases. The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.
- Subjects :
- Amino Acid Sequence
Animals
Autoantigens metabolism
Base Sequence
Cloning, Molecular
DNA
Escherichia coli genetics
Humans
Molecular Sequence Data
Protein Conformation
Sequence Homology, Amino Acid
Swine
beta-Galactosidase chemistry
beta-Galactosidase metabolism
Autoantigens immunology
Endothelium, Vascular immunology
Glycoside Hydrolases
Kidney blood supply
beta-Galactosidase genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 275
- Issue :
- 25
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 10858461
- Full Text :
- https://doi.org/10.1074/jbc.M001888200