Ogawa Haruko, Kobayashi Takaaki, Yokoyama Itsuo, Nagatani Naoki, Mizuno Masaaki, Yoshida Jun, Kadomatsu Kenji, Muramatsu Hisako, Nakao Akimasa, Muramatsu Takashi
Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan.
Xenotransplantation. 2002 Jul;9(4):290-6. doi: 10.1034/j.1399-3089.2002.01098.x.
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.
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.
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.
消除α-半乳糖（Galα1-3Galβ1-4GlcNAc，αGal）表位被认为是猪到人的异种移植成功的关键，但遗憾的是，这一目标尚未实现。β-半乳糖苷酶C（EndoGalC）是一种内切糖苷酶，可切割αGal表位中的Galβ1-4GlcNAc连接，并消化掉Galα1-3Gal二糖。由于其在生理pH条件下具有强大的活性，EndoGalC几乎可以完全去除猪红细胞和血管内皮细胞表面表达的αGal表位。体内或体外给予EndoGalC成功地将猪肾脏中的αGal表达降低到无法检测的水平，但αGal表位很快又重新出现。尝试在猪细胞中进行EndoGalC的基因表达以解决这一问题。由于末端αGal是由α-1,3-半乳糖基转移酶（α1,3GT）在反式高尔基体网络中转移的，因此EndoGalC基因与α1,3GT基因的共定位有望成为消除αGal表位最可靠的方法之一。
将猪α1,3GT的序列（包括胞质尾、跨膜结构域和茎区）连接到EndoGalC的上游，并在猪主动脉内皮细胞和COS7细胞中表达该共轭基因。导入该基因后，猪主动脉内皮细胞上的αGal表位得到有效减少。在COS7细胞中使用EndoGalC与α1,3GT进行转染研究表明，表达的EndoGalC不仅定位于细胞内，也定位于细胞外。与鼠免疫球蛋白（Igκ）链信号序列共轭的EndoGalC的表达也显示出类似的效果。
这些结果表明将EndoGalC基因转移到猪内皮细胞中的有效性，并强烈鼓励我们培育表达该酶的转基因动物。
