High-throughput screening of glycan-binding proteins using miniature pig kidney N-glycan-immobilized beads.
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.
Kim
Yun-Gon
YG
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Korea.
Shin
Dong-Sik
DS
Yang
Yung-Hun
YH
Gil
Geun-Cheol
GC
Park
Chung-Gyu
CG
Mimura
Yusuke
Y
Cooper
David K C
DK
Rudd
Pauline M
PM
Dwek
Raymond A
RA
Lee
Yoon-Sik
YS
Kim
Byung-Gee
BG
D000818:Animals; D001565:Benzoates / Q000378:metabolism; D006084:Graft Rejection; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D013058:Mass Spectrometry; D008863:Microspheres; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013556:Swine, Miniature; D014183:Transplantation, Heterologous / Q000276:immunology
D001565:Benzoates; D011134:Polysaccharides; D011506:Proteins; C053198:4-hydrazinobenzoic acid