Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.
Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.
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编码新的异种反应性抗原的猪β1,4N-乙酰乳糖苷转移酶的克隆和表达。
DOI：
10.1111/xen.12124
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发表时间：
2014-11
影响因子：
3.9
通讯作者：
McGregor CG
中科院分区：
文献类型：
--
作者：
Byrne GW;Du Z;Stalboerger P;Kogelberg H;McGregor CG
GGTA-1基因（GTKO）工程突变的猪器官异种移植排斥反应仍然是一个主要的抗体介导过程，该过程直接针对各种非半乳糖蛋白和碳水化合物抗原。我们之前使用表达文库筛选策略鉴定了6种猪内皮细胞cdna，它们编码猪抗原，与猪-灵长类动物心脏异种移植后诱导的IgG结合。其中一个基因产物是与牛β1,4 n -乙酰半乳糖氨基转移酶（B4GALNT2）同源的糖基转移酶。我们现在描述了猪B4GALNT2基因序列、基因组组织、表达和功能意义。从原始文库分离物中恢复猪B4GALNT2 cDNA，进行亚克隆、测序，并用于鉴定来自奥克兰儿童医院研究所BACPAC资源中心（#AC173453）的含有整个B4GALNT2位点的细菌人工染色体（BAC）。PCR引物用于绘制BAC克隆的内含子/外显子基因组组织。制备了表达猪B4GALNT2 （HEK- b4t）的人胚胎肾（HEK）稳定细胞系。猪B4GALNT2在HEK-B4T细胞中的表达通过免疫染色和siRNA转染进行了表征。采用流式细胞术和补体介导裂解法检测B4GALNT2对HEK-B4T细胞表达的影响。利用结合HEK和HEK- b4t细胞的抗体检测对B4GALNT2产生的聚糖的诱导抗体反应，并将结果与GTKO PAEC特异性非gal抗体诱导进行比较。采用定性和定量实时逆转录酶PCR和双花莲凝集素（DBA）组织染色法检测猪B4GALNT2在猪细胞和组织中的表达。猪B4GALNT2基因具有保守的基因组结构，其编码的开放阅读框与人类和小鼠B4GALNT2基因分别具有76%和70%的氨基酸同源性。B4GALNT2基因在猪内皮细胞中表达，具有广泛的分布表达模式。猪B4GALNT2在人HEK细胞中的表达（HEK- b4t）导致抗体与B4GALNT2酶的结合增加，并且与抗sda和DBA的反应性增强。猪到灵长类动物心脏异种移植后，HEK-B4T细胞对补体介导的溶解表现出更高的敏感性。在GTKO和GTKO:CD55心脏异种移植受者中，使用GTKO PAECs测量的非gal抗体的诱导与优先结合HEK-B4T细胞的抗体的诱导之间存在显著相关性。从PAEC表达文库中分离出猪B4GALNT2基因的功能，B4GALNT2基因的表达模式及其对HEK-B4T细胞对抗体结合和补体介导的酵解的致敏性表明，猪B4GALNT2的酶活性产生了一种新的免疫原性非半乳糖聚糖，这在一定程度上有助于猪-狒狒心脏异种移植后检测到的非半乳糖免疫应答。
Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation.
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