Electron microscopic and immunochemical analysis of the broadly neutralizing HIV-1-specific, anti-carbohydrate antibody, 2G12.
2G12 is one of only a few cloned antibodies with broadly neutralizing specificity to HIV-1 envelope proteins. Crystallographic and electron microscopic (EM) data showed that the Fab arms are locked together via a novel VH domain exchange. Both the conventional and the unprecedented additional VH-VH antigen binding sites show specificity for high mannose oligosaccharides on the silent face of gp120. We have now extended the EM and biochemical analysis of 2G12. Unligated 2G12 IgG1 molecules clearly show paired (parallel attached) Fab arms in the "doughnut" configuration attached to the Fc both in individual and computationally averaged images. A minority of the IgG molecules in the 2G12 prep showed the open "Y" configuration of conventional IgG. The averaged EM image compares well to the atomic structure model of 2G12. Papain digests of 2G12 yielded paired Fab arms (Fab dimer), as observed by EM, which dissociated into Fab-sized fragments in non-reducing SDS-PAGE. Purified 2G12 reduced and alkylated H and L chains can reassociate to form IgG molecules with the Fab dimer configuration and can combine with L and H chains from conventional human IgG to form hybrid molecules. 2G12 is heavily aggregated following brief acid exposure possibly as a result of its unique structure. A model of the aggregation process is proposed. An anti-Id MAb was shown by EM to react with neither the conventional nor additional antigen binding sites, but bound to the lateral faces of the Fab arms of intact, reduced and alkylated, and reconstructed 2G12 molecules. Efforts to identify IgG molecules with a similar intertwined Fab dimer structure in a large IgG pool were unsuccessful.
Department of Biological Science, 2nd Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4307, USA. kroux@bio.fsu.edu
K H
KH
Roux
Ping
P
Zhu
Margaret
M
Seavy
Hermann
H
Katinger
Renate
R
Kunert
Vanessa
V
Seamon
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000648:ultrastructure; D002241:Carbohydrates / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000276:immunology / Q000648:ultrastructure; D007150:Immunohistochemistry; D008854:Microscopy, Electron
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments