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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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Global shape of 2G12-IgM. (A) Experimental X-ray scattering data of IgMs. The s axis is shown in a logarithmic representation. (B) SAXS data showing a comparison of the experimental radial density distributions of IgMs. Calculated Rg values are shown. (C) Models of IgM-012, IgM-012_GL and IgM-617 (control) proteins and their overlay. Models were calculated by DAMMIF using an average of 50 structures, with P5 symmetry being imposed.
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f0025: Global shape of 2G12-IgM. (A) Experimental X-ray scattering data of IgMs. The s axis is shown in a logarithmic representation. (B) SAXS data showing a comparison of the experimental radial density distributions of IgMs. Calculated Rg values are shown. (C) Models of IgM-012, IgM-012_GL and IgM-617 (control) proteins and their overlay. Models were calculated by DAMMIF using an average of 50 structures, with P5 symmetry being imposed.

Mentions: For further structural evaluation small-angle X-ray scattering (SAXS) was employed. The raw data (Fig. 5A) already allows to clearly discriminate between IgM-617 and the two 2G12-IgM variants, for which almost identical scattering curves were obtained. This is further supported by the calculated gyration radius (Rg) values showing that IgM-012 and IgM-012_GL adopt a less compact fold than IgM-617 (Fig. 5B). Notably, the determined Rg values are in good agreement with those of previous SAXS experiments on IgMs [33,34]. A five-fold symmetry was imposed for low-resolution shape modeling of IgM-012 and IgM-617, and a total of 50 structures were used to generate density models. In agreement with the scattering curves and the pair distance distribution function, the superimposition of the shape models confirms that IgM-617 adopts a more compact conformation than IgM-012 and IgM-012_GL (Fig. 5C).


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)

Global shape of 2G12-IgM. (A) Experimental X-ray scattering data of IgMs. The s axis is shown in a logarithmic representation. (B) SAXS data showing a comparison of the experimental radial density distributions of IgMs. Calculated Rg values are shown. (C) Models of IgM-012, IgM-012_GL and IgM-617 (control) proteins and their overlay. Models were calculated by DAMMIF using an average of 50 structures, with P5 symmetry being imposed.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0025: Global shape of 2G12-IgM. (A) Experimental X-ray scattering data of IgMs. The s axis is shown in a logarithmic representation. (B) SAXS data showing a comparison of the experimental radial density distributions of IgMs. Calculated Rg values are shown. (C) Models of IgM-012, IgM-012_GL and IgM-617 (control) proteins and their overlay. Models were calculated by DAMMIF using an average of 50 structures, with P5 symmetry being imposed.
Mentions: For further structural evaluation small-angle X-ray scattering (SAXS) was employed. The raw data (Fig. 5A) already allows to clearly discriminate between IgM-617 and the two 2G12-IgM variants, for which almost identical scattering curves were obtained. This is further supported by the calculated gyration radius (Rg) values showing that IgM-012 and IgM-012_GL adopt a less compact fold than IgM-617 (Fig. 5B). Notably, the determined Rg values are in good agreement with those of previous SAXS experiments on IgMs [33,34]. A five-fold symmetry was imposed for low-resolution shape modeling of IgM-012 and IgM-617, and a total of 50 structures were used to generate density models. In agreement with the scattering curves and the pair distance distribution function, the superimposition of the shape models confirms that IgM-617 adopts a more compact conformation than IgM-012 and IgM-012_GL (Fig. 5C).

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