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抗受体

凝集素与聚糖的相互作用在许多生物系统中都起着重要的作用，但是人们对细胞膜上表达的糖蛋白反受体的性质却知之甚少。为了帮助解决此问题，我们开发了一种基于邻近标签技术的方法。使用过氧化物酶偶联的凝集素，添加H ₂ O ₂和酪酰胺-生物素底物会导致短程生物素自由基的产生，这些自由基会在结合的凝集素的紧邻处对蛋白质进行生物素化，随后可以对其进行鉴定。作为原理的证明，唾液酸粘蛋白-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反应和酪酰胺-SS-生物素。具有最强生物素化作用的红细胞膜蛋白被鉴定为糖蛋白A，这与使用凝集素覆盖和再糖基化方法的早期研究相一致。作为对该方法的进一步测试，将植物凝集素MAL II与辣根过氧化物酶偶联，并用于人类红细胞的邻近标记。再次对糖蛋白A进行了选择性标记，这与先前报道的MAL II对糖蛋白具有高亲和力相一致。该方法可以应用于其他凝集素以鉴定其膜反受体。