Design, synthesis and characterisation of affinity ligands for glycoproteins.
The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein-carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar-binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine-based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself.
Institute of Biotechnology, University of Cambridge, Tennis Court Rd, Cambridge, CB2 1QT, UK.
U D
UD
Palanisamy
A
A
Hussain
S
S
Iqbal
K
K
Sproule
C R
CR
Lowe
D000327:Adsorption; D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D020650:Combinatorial Chemistry Techniques; D015195:Drug Design; D005656:Fungal Proteins / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D008024:Ligands; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013329:Structure-Activity Relationship
D000345:Affinity Labels; D005656:Fungal Proteins; D006023:Glycoproteins; D008024:Ligands; D009844:Oligosaccharides; D005949:Glucose Oxidase; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic