Miwa Yuko, Kobayashi Takaaki, Nagasaka Takaharu, Liu DaGe, Yu Ma, Yokoyama Itsuo, Suzuki Akemi, Nakao Akimasa
Department of Surgery II, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Xenotransplantation. 2004 May;11(3):247-53. doi: 10.1111/j.1399-3089.2004.00126.x.
N-glycolylneuraminic acid (NeuGc) epitopes, so called Hanganutziu-Deicher (HD) antigens, which are widely expressed on endothelial cells of all mammals except humans, are considered to be potential targets for natural and elicited anti-nonGalalpha1-3 Gal (Gal) antibodies in humans. We previously reported that anti-NeuGc antibodies were not detected in healthy humans by enzyme-linked immunosorbent assay (ELISA) using NeuGc-GM3-coated plates, and no antibody production was observed in patients with a history of exposure to pig cells. However, a recent paper has revealed that (i) anti-NeuGc antibodies to porcine red blood cells (PRBC) are detectable in most healthy humans, and (ii) the majority of anti-nonGal antibodies are specific for NeuGc epitopes. The purpose of this study was to reassess whether NeuGc is important as an immunogenic nonGal epitope.
The binding of antibodies to PRBC and porcine aortic endothelial cells (PAEC) was compared. Cells were treated with (i) alpha-galactosidase, and then (ii) neuraminidase, which digests sialic acids, including NeuGc epitopes. Cells were incubated with human pooled sera, and applied to flow cytometric analysis. After enzyme digestion, almost complete reduction of Gal and NeuGc expression was confirmed by GS-IB4 and HU/Ch2-7 (a chicken monoclonal antibody against HD antigens), respectively. Trypsin, which removes membrane glycoproteins, and endoglycoceramidase which cleaves glycolipids, were used for differentiating between NeuGc-containing glycoproteins and glycolipids.
Neuraminidase-treatment reduced the binding of immunoglobulin G (IgG) antibodies to PRBC; about half of the anti-nonGal IgG antibodies to PRBC were directed to NeuGc. In contrast, anti-nonGal antibodies to PAEC were not directed to NeuGc. Trypsin-treatment markedly reduced the expression of NeuGc only on PRBC. Endoglycoceramidase-treatment was followed by a greater reduction in NeuGc epitopes on PAEC than on PRBC. Most NeuGc on PRBC appeared to be linked to proteins, but most NeuGc on PAEC was expressed on glycolipids.
Carbohydrate structures on PRBC are different from those on PAEC. Healthy human sera contain anti-nonGal IgG antibodies to NeuGc expressed on PRBC, but not on PAEC. We speculate that anti-nonGal IgG antibodies to PRBC can recognize both NeuGc and protein, and this may be the reason why such antibodies have not been detected by ELISA. A definite conclusion about the immunogenicity of NeuGc has not been obtained. More sera from patients (not from non-human primates) sensitized with porcine cells or organs need to be studied.
N-羟乙酰神经氨酸（NeuGc）表位，即所谓的汉加纽齐乌-戴歇尔（HD）抗原，广泛表达于除人类以外的所有哺乳动物的内皮细胞上，被认为是人类天然和诱导产生的抗非Galα1-3Gal（Gal）抗体的潜在靶点。我们之前报道，使用包被有NeuGc-GM3的酶联免疫吸附测定（ELISA）在健康人体内未检测到抗NeuGc抗体，且有猪细胞接触史的患者未观察到抗体产生。然而，最近一篇论文显示：（i）在大多数健康人体内可检测到针对猪红细胞（PRBC）的抗NeuGc抗体；（ii）大多数抗非Gal抗体对NeuGc表位具有特异性。本研究的目的是重新评估NeuGc作为一种免疫原性非Gal表位是否重要。
比较抗体与PRBC和猪主动脉内皮细胞（PAEC）的结合情况。细胞先经（i）α-半乳糖苷酶处理，然后经（ii）神经氨酸酶处理，神经氨酸酶可消化包括NeuGc表位在内的唾液酸。细胞与人混合血清孵育后，进行流式细胞术分析。酶消化后，分别通过GS-IB4和HU/Ch2-7（一种抗HD抗原的鸡单克隆抗体）确认Gal和NeuGc表达几乎完全降低。使用胰蛋白酶去除膜糖蛋白，使用内切糖神经酰胺酶切割糖脂，以区分含NeuGc的糖蛋白和糖脂。
神经氨酸酶处理降低了免疫球蛋白G（IgG）抗体与PRBC的结合；约一半针对PRBC的抗非Gal IgG抗体针对NeuGc。相反，针对PAEC的抗非Gal抗体不针对NeuGc。胰蛋白酶处理仅显著降低了PRBC上NeuGc的表达。内切糖神经酰胺酶处理后，PAEC上NeuGc表位的减少比PRBC上更明显。PRBC上的大多数NeuGc似乎与蛋白质相连，但PAEC上的大多数NeuGc表达于糖脂上。
PRBC上的碳水化合物结构与PAEC上的不同。健康人血清中含有针对PRBC上表达的NeuGc而非PAEC上表达的NeuGc的抗非Gal IgG抗体。我们推测针对PRBC的抗非Gal IgG抗体可识别NeuGc和蛋白质，这可能是ELISA未检测到此类抗体的原因。尚未得出关于NeuGc免疫原性的确切结论。需要研究更多来自猪细胞或器官致敏患者（而非非人灵长类动物）的血清。
