In situ visualization of a glycoform of transferrin: localization of α2,6-sialylated transferrin in the liver
In situ visualization of a glycoform of transferrin: localization of α2,6-sialylated transferrin in the liver
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DOI：
10.1093/jb/mvu071
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发表时间：
2015-04-01
影响因子：
2.7
通讯作者：
Hashimoto, Yasuhiro
中科院分区：
文献类型：
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作者：
Matsumoto, Yuka;Saito, Toshie;Hashimoto, Yasuhiro
我们先前发现一种凝集素，接骨木凝集素(SSA)，与转铁蛋白上的α-2，6-唾液酸化的糖链表位结合，并抑制抗转铁蛋白抗体与抗原的结合(SSA抑制)。在这里，我们报告SSA抑制适用于免疫组织化学，定位肝脏中的α2，6-唾液酸化转铁蛋白。用抗转铁蛋白多克隆抗体进行免疫组织化学染色，发现转铁蛋白位于小叶间静脉附近的肝细胞中。加入SSA凝集素后，染色明显减弱。唾液酸酶处理的肝脏切片取消了SSA结合，同时取消了SSA抑制，这表明SSA与该切片上的糖链表位的结合是抑制作用的关键。为了检验抗原表位和SSA结合(糖基化)位点之间接近的重要性，我们制备了两种针对转铁蛋白部分氨基酸序列的抗肽抗体。一种抗体(Tf-596AB)是针对N-糖基化位点(ASN-432和ASN-630)附近的肽序列Cys596-Ala614。另一种(Tf-120Ab)是针对这些位点远端的一个肽序列Val120-Cys137。加入SSA后，TF-596AB的染色信号减少，而TF-120Ab的染色信号仅略有减少。这一结果表明，抗原表位与SSA结合部位的接近是免疫组织化学中抑制SSA的关键。
We previously found that a lectin, Sambucus sieboldiana agglutinin (SSA), bound to alpha 2,6-sialylated glycan epitopes on transferrin and inhibited anti-transferrin antibody binding to the antigen in ELISA (SSA inhibition). Here we report that SSA inhibition is applicable to immunohistochemistry, localizing alpha 2,6-sialylated transferrin in the liver. Immunohistochemistry using anti-transferrin polyclonal antibody revealed that transferrin was detected in hepatocytes near interlobular veins. Addition of SSA lectin markedly attenuated the staining. Sialidase treatment of a liver section abolished SSA binding and concomitantly cancelled SSA inhibition, suggesting that SSA binding to glycan epitopes on the section was essential for the inhibition. To examine the importance of proximity between antigen epitopes and SSA-binding (glycosylation) sites, we prepared two anti-peptide antibodies against partial amino acid sequences of transferrin. One antibody (Tf-596Ab) is against a peptide sequence, Cys596-Ala614, which is proximal to N-glycosylation sites (Asn-432 and Asn-630). The other (Tf-120Ab) is against a peptide sequence, Val120-Cys137, distal to the sites. The staining signals of Tf-596Ab were reduced by the addition of SSA, whereas those of Tf-120Ab were reduced only a little. This result suggests that proximity of the antigen epitope to SSA binding sites is critical for SSA inhibition in immunohistochemistry.