High-throughput screening of glycan-binding proteins using miniature pig kidney N-glycan-immobilized beads.

Yun-Gon Kim, Dong-Sik Shin, Yung-Hun Yang, Geun-Cheol Gil, Chung-Gyu Park, Yusuke Mimura, David K C Cooper, Pauline M Rudd, Raymond A Dwek, Yoon-Sik Lee, Byung-Gee Kim

Index: Chem. Biol. 15(3) , 215-23, (2008)

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Abstract

Glycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.