Avilés M, Castells M T, Martínez-Menárguez J A, Abascal I, Ballesta J
Department of Cell Biology, Medical School, University of Murcia, Spain.
Histochem J. 1997 Aug;29(8):583-92. doi: 10.1023/a:1026432211012.
Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase-PNA/SBA and galactose oxidase-neuraminidase-PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins.
来自花生的凝集素（PNA）和大豆的凝集素（SBA）分别结合半乳糖（Gal）和N-乙酰半乳糖胺（GalNAc）的末端残基。半乳糖氧化酶氧化末端Gal和GalNAc的C-6位羟基，从而阻断PNA和SBA的结合。唾液酸末端残基的存在也会严重限制这些凝集素与糖残基的结合。在本研究中，凝集素细胞化学结合酶处理和定量分析已应用于光镜和电镜水平，以开发一种简单的方法，能够在原位区分倒数第二个和末端的Gal/GalNAc残基。用于该方法演示的选定区域包括大鼠透明带和大鼠精子细胞的顶体，它们含有丰富的带有末端Gal/GalNAc残基的糖蛋白。透明带用LFA、PNA和SBA标记。半乳糖氧化酶处理后，末端Gal/GalNAc残基被氧化，对PNA/SBA的反应性消失。依次应用半乳糖氧化酶、神经氨酸酶和PNA/SBA会产生以下效果：（i）末端Gal/GalNAc残基的氧化；（ii）末端唾液酸残基的去除，使凝集素能够结合倒数第二个Gal/GalNAc残基；（iii）凝集素与糖残基的结合。顶体对PNA和SBA有反应。未检测到LFA反应性，因此表明不存在末端唾液酸残基。因此，在半乳糖氧化酶-PNA/SBA和半乳糖氧化酶-神经氨酸酶-PNA/SBA序列处理后均未观察到标记。总之，半乳糖氧化酶、神经氨酸酶和PNA/SBA细胞化学的联合应用是一种用于显示对这些凝集素有亲和力的倒数第二个碳水化合物残基的有用技术。
