Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequence alignment and structural model of 2G12-IgM. A) Alignment of IgM-012 and the respective germline sequences IGHV3-21/JH3 and IGKV1-5/JK1 using Clustal 2.0.8. Sequences are numbered according to the Kabat system. Complementarity-determining regions are in bold letters. Canonical residues are underlined and Vernier residues are marked by asterisks. Residues previously identified as structurally and functionally important [32,34] are displayed in red. (B) Superimposition of 2G12-IgG (PDB identifier: 1OM3) with models of IgM-012_GL heavy and light chains. The introduced germline residues and their wild-type counterparts are displayed in blue and red, respectively. Note that in the model of the IgM-012_GL heavy chain, the CH1 domain does not overlap with that of 2G12-IgG due to differences in the orientation of a flexible loop.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582045&req=5

f0005: Sequence alignment and structural model of 2G12-IgM. A) Alignment of IgM-012 and the respective germline sequences IGHV3-21/JH3 and IGKV1-5/JK1 using Clustal 2.0.8. Sequences are numbered according to the Kabat system. Complementarity-determining regions are in bold letters. Canonical residues are underlined and Vernier residues are marked by asterisks. Residues previously identified as structurally and functionally important [32,34] are displayed in red. (B) Superimposition of 2G12-IgG (PDB identifier: 1OM3) with models of IgM-012_GL heavy and light chains. The introduced germline residues and their wild-type counterparts are displayed in blue and red, respectively. Note that in the model of the IgM-012_GL heavy chain, the CH1 domain does not overlap with that of 2G12-IgG due to differences in the orientation of a flexible loop.

Mentions: The rational design of IgM-012_GL was based on the primary sequence of IgM-012. Ten mutations were introduced into the framework of IgM-012 vH and 7 mutations into that of IgM-012 vL (Tables 1a and 1b). In essence, all residues were changed into their germline counterparts except those which are (i) known to be important for antigen binding (as identified by previous studies), (ii) located in Vernier zones, and (iii) responsible for the unique domain swap of 2G12 (Fig. 1, Tables 1a and 1b). While the percentage of identities between IgM-012 and the corresponding germline sequences is 68% (vH) and 84% (vL), these values increase to 78% and 91% in the case of IgM-012_GL, leading to an improvement of the germinality index [31] from 0.8 (IgM-012) to 0.9 (IgM-012_GL). However, the natural IgM-617 displays an even higher extent of homology with its germline counterpart (vH: 95%; vL: 98%). It should be noted that the mutations introduced into IgM-012 did not lead to substantial changes in hydrophobicity (IgM-012: 244 hydrophobic residues; IgM-012_GL: 243 hydrophobic residues; total number of residues in both cases: 789) and isoelectric point (IgM-012: 6.72; IgM-012_GL: 6.81).


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)

Sequence alignment and structural model of 2G12-IgM. A) Alignment of IgM-012 and the respective germline sequences IGHV3-21/JH3 and IGKV1-5/JK1 using Clustal 2.0.8. Sequences are numbered according to the Kabat system. Complementarity-determining regions are in bold letters. Canonical residues are underlined and Vernier residues are marked by asterisks. Residues previously identified as structurally and functionally important [32,34] are displayed in red. (B) Superimposition of 2G12-IgG (PDB identifier: 1OM3) with models of IgM-012_GL heavy and light chains. The introduced germline residues and their wild-type counterparts are displayed in blue and red, respectively. Note that in the model of the IgM-012_GL heavy chain, the CH1 domain does not overlap with that of 2G12-IgG due to differences in the orientation of a flexible loop.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0005: Sequence alignment and structural model of 2G12-IgM. A) Alignment of IgM-012 and the respective germline sequences IGHV3-21/JH3 and IGKV1-5/JK1 using Clustal 2.0.8. Sequences are numbered according to the Kabat system. Complementarity-determining regions are in bold letters. Canonical residues are underlined and Vernier residues are marked by asterisks. Residues previously identified as structurally and functionally important [32,34] are displayed in red. (B) Superimposition of 2G12-IgG (PDB identifier: 1OM3) with models of IgM-012_GL heavy and light chains. The introduced germline residues and their wild-type counterparts are displayed in blue and red, respectively. Note that in the model of the IgM-012_GL heavy chain, the CH1 domain does not overlap with that of 2G12-IgG due to differences in the orientation of a flexible loop.
Mentions: The rational design of IgM-012_GL was based on the primary sequence of IgM-012. Ten mutations were introduced into the framework of IgM-012 vH and 7 mutations into that of IgM-012 vL (Tables 1a and 1b). In essence, all residues were changed into their germline counterparts except those which are (i) known to be important for antigen binding (as identified by previous studies), (ii) located in Vernier zones, and (iii) responsible for the unique domain swap of 2G12 (Fig. 1, Tables 1a and 1b). While the percentage of identities between IgM-012 and the corresponding germline sequences is 68% (vH) and 84% (vL), these values increase to 78% and 91% in the case of IgM-012_GL, leading to an improvement of the germinality index [31] from 0.8 (IgM-012) to 0.9 (IgM-012_GL). However, the natural IgM-617 displays an even higher extent of homology with its germline counterpart (vH: 95%; vL: 98%). It should be noted that the mutations introduced into IgM-012 did not lead to substantial changes in hydrophobicity (IgM-012: 244 hydrophobic residues; IgM-012_GL: 243 hydrophobic residues; total number of residues in both cases: 789) and isoelectric point (IgM-012: 6.72; IgM-012_GL: 6.81).

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