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 labelling 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 reacte... More
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 labelling 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 labelling of human erythrocytes. Glycophorin A
was again selectively labelled, 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.
