Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney.
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.
Ogawa
H
H
Department of Biochemistry and Department of Surgery II, Nagoya 466-8550, Japan.
Muramatsu
H
H
Kobayashi
T
T
Morozumi
K
K
Yokoyama
I
I
Kurosawa
N
N
Nakao
A
A
Muramatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004730:Endothelium, Vascular / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases; D006801:Humans; D007668:Kidney / Q000098:blood supply*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013552:Swine; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001324:Autoantigens; D004247:DNA; D006026:Glycoside Hydrolases; C057030:endo-beta-D-galactosidase C; D001616:beta-Galactosidase