Lectin–glycan interactions play important roles in many biological systems, but the nature of glycoprotein counter-receptors expressed on cell membranes is often poorly understood. To help overcome this problem, we developed a method based on proximity labeling technology. Using a peroxidase-coupled lectin, addition of H₂O₂ and tyramide-biotin substrates leads to generation of short-range biotin radicals that biotinylate proteins in the immediate vicinity of the bound lectin, which can subsequently be identified. As a proof-of-principle, sialoadhesin-horseradish peroxidase-human IgG1 Fc recombinant protein constructs were precomplexed with anti-Fc antibodies, bound to human erythrocytes and reacted with H₂O₂ and tyramide-SS-biotin. The erythrocyte membrane protein with strongest biotinylation was identified as glycophorin A, in agreement with early studies using lectin overlay and reglycosylation approaches. As a further test of the method, the plant lectin MAL II was conjugated with horseradish peroxidase and used in proximity labeling of human erythrocytes. Glycophorin A was again selectively labeled, which is consistent with previous reports that MAL II has high affinity for glycophorin. This method could be applied to other lectins to identify their membrane counter-receptors.

中文翻译：

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通过邻近标记鉴定细胞膜上的凝集素反受体

凝集素-聚糖相互作用在许多生物系统中发挥着重要作用，但人们对细胞膜上表达的糖蛋白反受体的性质通常知之甚少。为了帮助克服这个问题，我们开发了一种基于邻近标记技术的方法。使用过氧化物酶偶联的凝集素，添加 H ₂ O ₂和酪酰胺-生物素底物会产生短程生物素自由基，使紧邻结合的凝集素的蛋白质生物素化，随后可以对其进行鉴定。作为原理验证，唾液酸粘附素-辣根过氧化物酶-人 IgG1 Fc 重组蛋白构建体与抗 Fc 抗体预复合，与人红细胞结合并与 H 2 _O 2_和酪酰胺-SS-生物素反应。生物素化最强的红细胞膜蛋白被鉴定为血型糖蛋白 A，这与使用凝集素覆盖和重新糖基化方法的早期研究一致。作为该方法的进一步测试，植物凝集素 MAL II 与辣根过氧化物酶缀合并用于人红细胞的邻近标记。血型糖蛋白 A 再次被选择性标记，这与之前的报道一致，即 MAL II 对血型糖蛋白具有高亲和力。该方法可应用于其他凝集素以鉴定其膜反受体。