Photoactivatable glycopolymers for the proteome-wide identification of fucose-α(1-2)-galactose binding proteins

Abstract

Although fucose-α(1-2)-galactose (Fucα(1-2)Gal)-containing glycans have been implicated in cognitive processes such as learning and memory, their precise molecular mechanisms are poorly understood. Here we employed the use of multivalent glycopolymers to enable the first proteome-wide identification of weak affinity, low abundance Fucα(1-2)Gal glycan-binding proteins (GBPs). Biotin-terminated glycopolymers containing photoactivatable cross-linking groups were designed to capture and enrich GBPs from rat brain lysates. Candidate proteins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for the synaptic vesicle protein SV2a using a knockout mouse system. The results suggest a role for SV2a-Fucα(1-2)Gal interactions in SV2a trafficking and synaptic vesicle recycling. More broadly, our studies outline a general chemical approach for the systems-level discovery of novel GBPs.

Figure 1

Strategy for the systems-level, proteome-wide identification of GBPs.

Scheme 1. Synthesis of Biotin-Functionalized, Photoactivatable Glycopolymers

Figure 2

(A) Selective capture of UEAI by 10. (B) Representative silver-stained gel of GBPs isolated from synaptosomal and membrane fractions. (C) Relative binding profiles of candidate GBPs (n = 3).

Figure 3

(A) Fucα(1-2)Gal expression on glycoproteins in FUT1^–/– and FUT2^–/– mice. (B) Enrichment of SV2a in the membrane fraction of FUT2^–/– mice (n = 3, *P < 0.02). (C) Increased colocalization (yellow) of SV2a and syt1 puncta along neuronal processes in FUT2^–/– cortical neurons (n = 70–80, **P < 0.005). (D) Increased cell surface biotinylation of SV2a in FUT2^–/– cortical neurons (n = 4, *** P < 0.05). See SI for experimental details.