Reduction of alpha-galactosyl xenoantigen by expression of endo-beta-galactosidase C in pig endothelial cells.
BACKGROUND
Elimination of the Galalpha1-3Galbeta1-4GlcNAc (alphaGal) epitope has been considered to be essential for successful pig-to-human xenotransplantation but, unfortunately, has not been achieved. Endo-beta-galactosidase C (EndoGalC) is an endoglycosidase which cleaves the Galbeta1-4GlcNAc linkage in the alphaGal epitope and digests out the Galalpha1-3Gal disaccharide. Because of its potent activity in physiological pH conditions, EndoGalC can remove alphaGal epitopes expressed on the cell surface of pig erythrocytes and vascular endothelial cells almost completely. In vivo or ex vivo administration of EndoGalC successfully reduced alphaGal expression in pig kidneys to an undetectable level, but alphaGal epitopes soon reappeared. Gene expression of EndoGalC in pig cells was attempted to solve this problem. As the terminal alphaGal is transferred in the trans-Golgi network by alpha-1,3-galactosyltransferase (alpha1,3GT), colocalization of the EndoGalC gene with the alpha1,3GT gene was expected to be one of the most reliable ways to eliminate the alphaGal epitope.


RESULTS
The sequence of pig alpha1,3GT, including the cytoplasmic tail, transmembrane domain and stem region, was ligated upstream of EndoGalC, and the conjugated gene was expressed in pig aortic endothelial cells and COS7 cells. Following the introduction of the gene, the alphaGal epitope on pig aortic endothelial cells was effectively reduced. Transfection studies in COS7 cells using EndoGalC combined with alpha1,3GT showed that the expressed EndoGalC was localized not only inside, but also outside, the cells. The expression of EndoGalC conjugated with a murine immunoglobulin (Igkappa)-chain signal sequence also showed a similar effect.


CONCLUSIONS
These results suggest the effectiveness of gene transfer with EndoGalC into pig endothelial cells, and strongly encourage us to produce transgenic animals with the expressed enzyme.
Ogawa
Haruko
H
Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan.
Kobayashi
Takaaki
T
Yokoyama
Itsuo
I
Nagatani
Naoki
N
Mizuno
Masaaki
M
Yoshida
Jun
J
Kadomatsu
Kenji
K
Muramatsu
Hisako
H
Nakao
Akimasa
A
Muramatsu
Takashi
T
D000818:Animals; D015478:Antigens, Heterophile / Q000276:immunology*; D001483:Base Sequence; D019556:COS Cells; D002240:Carbohydrate Sequence; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004187:Disaccharides / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000276:immunology*; D000939:Epitopes / Q000276:immunology*; D006026:Glycoside Hydrolases; D007668:Kidney / Q000276:immunology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014312:Trisaccharides / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism*
D015478:Antigens, Heterophile; D017931:DNA Primers; D004187:Disaccharides; D000939:Epitopes; D011994:Recombinant Proteins; D014312:Trisaccharides; C055075:galactosyl-(1-3)galactose; D006026:Glycoside Hydrolases; C057030:endo-beta-D-galactosidase C; D001616:beta-Galactosidase