科研之友
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摘要
Antibodies with conformational specificity are important for detecting and interfering with polypeptide aggregation linked to several human disorders. We are developing a motif-grafting approach for designing lead antibody candidates specific for amyloid-forming polypeptides such as the Alzheimer peptide (A beta). This approach involves grafting amyloidogenic peptide segments into the complementarity-determining regions (CDRs) of single-domain (V-H) antibodies. Here we have investigated the impact of polar mutations inserted at the edges of a large hydrophobic A beta 42 peptide segment (A beta residues 17-42) in CDR3 on the solubility and conformational specificity of the corresponding V-H domains. We find that V-H expression and solubility are strongly enhanced by introducing multiple negatively charged or asparagine residues at the edges of CDR3, whereas other polar mutations are less effective (glutamine and serine) or ineffective (threonine, lysine, and arginine). Moreover, A beta V-H domains with negatively charged CDR3 mutations show significant preference for recognizing A beta fibrils relative to A beta monomers, whereas the same V-H domains with other polar CDR3 mutations recognize both A beta conformers. We observe similar behavior for a V-H domain grafted with a large hydrophobic peptide from islet amyloid polypeptide (residues 8-37) that contains negatively charged mutations at the edges of CDR3. These findings highlight the sensitivity of antibody binding and solubility to residues at the edges of CDRs, and provide guidelines for designing other grafted antibody fragments with hydrophobic binding loops.
- 出版日期2016-2-5
