Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.
Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.Show MeSH
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Mentions: Finally, the conformational stability was probed by chemical denaturation of the four recombinant antibodies using increasing concentrations of urea (0–8 M). The change in tryptophan fluorescence intensity was monitored spectrofluorimetrically. Fig. 4 depicts the corresponding curves demonstrating that 2G12-IgG is stable up to 5 M urea. Unfolding starts at 5.5 M urea, but does not reach completion even at the highest urea concentration used (8 M). Its IgM counterpart IgM-012 already shows some minor unfolding between 1 and 4 M urea, but this is again followed by a steady increase in fluorescence without achieving an upper plateau. The presence of germline residues in IgM-012_GL only slightly increased its chemical stability, with the first (minor) transition being less pronounced than in the case of its wild-type counterpart. These data are comparable to natural IgM-617 that also follows a non-two-state transition with a first (minor) unfolding event below 3 M and a second (major) one within the range of 4–7 M urea (Fig. 4).
Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.