Osman N, McKenzie I F, Ostenried K, Ioannou Y A, Desnick R J, Sandrin M S
Molecular Immunogenetics Laboratory, Austin Research Institute, Austin Hospital, Heidelberg Victoria 3084 Australia.
Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14677-82. doi: 10.1073/pnas.94.26.14677.
Hyperacute rejection of pig organs by humans involves the interaction of Galalpha(1,3)Gal with antibodies and complement. Strategies to reduce the amount of xenoantigen Galalpha(1,3)Gal were investigated by overexpression of human lysosomal alpha-galactosidase in cultured porcine cells and transgenic mice. The overexpression of human alpha-galactosidase in cultured porcine endothelial cells and COS cells resulted in a 30-fold reduction of cell surface Galalpha(1,3)Gal and a 10-fold reduction in cell reactivity with natural human antibodies. Splenocytes from transgenic mice overexpressing human alpha-galactosidase showed only a 15-25% reduction in binding to natural human anti-Galalpha(1,3)Gal antibodies; however, this decrease was functionally significant as demonstrated by reduced susceptibility to human antibody-mediated lysis. However, because there is residual Galalpha(1,3)Gal and degalactosylation results in the exposure of N-acetyllactosamine residues and potential new xenoepitopes, using alpha-galactosidase alone is unlikely to overcome hyperacute rejection. We previously reported that mice overexpressing human alpha1,2-fucosyltransferase as a transgene had approximately 90% reduced Galalpha(1,3)Gal levels due to masking of the xenoantigen by fucosylation; we evaluated the effect of overexpressing alpha-galactosidase and alpha1,2-fucosyltransferase on Galalpha(1,3)Gal levels. Galalpha(1, 3)Gal-positive COS cells expressing alpha1,3-galactosyltransferase, alpha1,2-fucosyltransferase, and alpha-galactosidase showed negligible cell surface staining and were not susceptible to lysis by human serum containing antibody and complement. Thus, alpha1, 2-fucosyltransferase and alpha-galactosidase effectively reduced the expression of Galalpha(1,3)Gal on the cell surface and could be used to produce transgenic pigs with negligible levels of cell surface Galalpha(1,3)Gal, thereby having no reactivity with human serum and improving graft survival.
人类对猪器官的超急性排斥反应涉及Galα(1,3)Gal与抗体及补体的相互作用。通过在培养的猪细胞和转基因小鼠中过表达人溶酶体α-半乳糖苷酶，研究了减少异种抗原Galα(1,3)Gal量的策略。在培养的猪内皮细胞和COS细胞中过表达人α-半乳糖苷酶，导致细胞表面Galα(1,3)Gal减少30倍，与天然人抗体的细胞反应性降低10倍。过表达人α-半乳糖苷酶的转基因小鼠的脾细胞与天然人抗Galα(1,3)Gal抗体的结合仅减少15%-25%；然而，如对人抗体介导的裂解敏感性降低所证明，这种减少在功能上具有重要意义。然而，由于存在残留的Galα(1,3)Gal，去半乳糖基化会导致N-乙酰乳糖胺残基和潜在新异种抗原表位的暴露，仅使用α-半乳糖苷酶不太可能克服超急性排斥反应。我们之前报道过，作为转基因过表达人α1,2-岩藻糖基转移酶的小鼠，由于岩藻糖基化对异种抗原的掩盖作用，其Galα(1,3)Gal水平降低了约90%；我们评估了过表达α-半乳糖苷酶和α1,2-岩藻糖基转移酶对Galα(1,3)Gal水平的影响。表达α1,3-半乳糖基转移酶、α1,2-岩藻糖基转移酶和α-半乳糖苷酶的Galα(1,3)Gal阳性COS细胞显示出可忽略不计 的细胞表面染色，并且不易被含抗体和补体的人血清裂解。因此，α1,2-岩藻糖基转移酶和α-半乳糖苷酶有效地降低了细胞表面Galα(1,3)Gal的表达，可用于培育细胞表面Galα(1,3)Gal水平可忽略不计的转基因猪，从而使其与人类血清无反应性并提高移植物存活率。
