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Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

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Temperature-mediated unfolding of 2G12 variants. A) Electronic circular dichroism spectroscopy. Far UV-spectra of 2G12-IgG, IgM-012, IgM-012_GL, and IgM-617 (control) in the native state (continuous lines) and in the completely unfolded state (at 80 °C; dashed lines). Temperature-mediated unfolding was followed at 218 nm. Insets display the corresponding van't Hoff plots. B) Differential scanning calorimetry. Thermograms are depicted with fits (red line) based on the assumption of non-two-state (i.e. multi-step) transitions. The ratio ΔHv/ΔH gives information about the cooperativity of a transition (ΔHv/ΔH < 1, non-cooperative; ΔHv/ΔH > 1, cooperative unfolding).
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f0015: Temperature-mediated unfolding of 2G12 variants. A) Electronic circular dichroism spectroscopy. Far UV-spectra of 2G12-IgG, IgM-012, IgM-012_GL, and IgM-617 (control) in the native state (continuous lines) and in the completely unfolded state (at 80 °C; dashed lines). Temperature-mediated unfolding was followed at 218 nm. Insets display the corresponding van't Hoff plots. B) Differential scanning calorimetry. Thermograms are depicted with fits (red line) based on the assumption of non-two-state (i.e. multi-step) transitions. The ratio ΔHv/ΔH gives information about the cooperativity of a transition (ΔHv/ΔH < 1, non-cooperative; ΔHv/ΔH > 1, cooperative unfolding).

Mentions: Next we probed the overall secondary structure and thermal stability of 2G12-IgG, IgM-012, IgM-012_GL and a natural IgM (IgM-617) by ECD spectroscopy. The far-UV spectra of these proteins were similar, exhibiting a minimum at 218 nm, which is typical for protein structures dominated by β-sheets (Fig. 3A left panel). Upon increasing the temperature to > 80 °C, the proteins unfolded irreversibly leading to far-UV spectra of increased ellipticity with a broad minimum between 205 and 220 nm (dashed lines in Fig. 3A, left panel). This indicates the residual presence of undefined structural elements after thermal denaturation of the antibodies, which agrees well with the observation that protein precipitation did not occur. Similar results were previously reported for antibodies of different isotypes [32].


Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

Temperature-mediated unfolding of 2G12 variants. A) Electronic circular dichroism spectroscopy. Far UV-spectra of 2G12-IgG, IgM-012, IgM-012_GL, and IgM-617 (control) in the native state (continuous lines) and in the completely unfolded state (at 80 °C; dashed lines). Temperature-mediated unfolding was followed at 218 nm. Insets display the corresponding van't Hoff plots. B) Differential scanning calorimetry. Thermograms are depicted with fits (red line) based on the assumption of non-two-state (i.e. multi-step) transitions. The ratio ΔHv/ΔH gives information about the cooperativity of a transition (ΔHv/ΔH < 1, non-cooperative; ΔHv/ΔH > 1, cooperative unfolding).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4582045&req=5

f0015: Temperature-mediated unfolding of 2G12 variants. A) Electronic circular dichroism spectroscopy. Far UV-spectra of 2G12-IgG, IgM-012, IgM-012_GL, and IgM-617 (control) in the native state (continuous lines) and in the completely unfolded state (at 80 °C; dashed lines). Temperature-mediated unfolding was followed at 218 nm. Insets display the corresponding van't Hoff plots. B) Differential scanning calorimetry. Thermograms are depicted with fits (red line) based on the assumption of non-two-state (i.e. multi-step) transitions. The ratio ΔHv/ΔH gives information about the cooperativity of a transition (ΔHv/ΔH < 1, non-cooperative; ΔHv/ΔH > 1, cooperative unfolding).
Mentions: Next we probed the overall secondary structure and thermal stability of 2G12-IgG, IgM-012, IgM-012_GL and a natural IgM (IgM-617) by ECD spectroscopy. The far-UV spectra of these proteins were similar, exhibiting a minimum at 218 nm, which is typical for protein structures dominated by β-sheets (Fig. 3A left panel). Upon increasing the temperature to > 80 °C, the proteins unfolded irreversibly leading to far-UV spectra of increased ellipticity with a broad minimum between 205 and 220 nm (dashed lines in Fig. 3A, left panel). This indicates the residual presence of undefined structural elements after thermal denaturation of the antibodies, which agrees well with the observation that protein precipitation did not occur. Similar results were previously reported for antibodies of different isotypes [32].

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH
Related in: MedlinePlus