Dataset Information

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

ABSTRACT: Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. 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.

SUBMITTER: Chromikova V

PROVIDER: S-EPMC4582045 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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

Chromikova Veronika V Mader Alexander A Hofbauer Stefan S Göbl Christoph C Madl Tobias T Gach Johannes S JS Bauernfried Stefan S Furtmüller Paul G PG Forthal Donald N DN Mach Lukas L Obinger Christian C Kunert Renate R

Biochimica et biophysica acta 20150305 10 Pt A

Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of thes ...[more]

PMID: 25748881

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Project description:While native blood group A-like glycans have not been demonstrated in prokaryotic microorganisms as a source of human "natural" anti-A isoagglutinin production, and metazoan eukaryotic N-acetylgalactosamine O-glycosylation of serine or threonine residues (O-GalNAc-Ser/Thr-R) does not occur in bacteria, the O-GalNAc glycan-bearing ovarian glycolipids, discovered in C57BL/10 mice, are complementary to the syngeneic anti-A-reactive immunoglobulin M (IgM), which is not present in animals that have undergone ovariectomy prior to the onset of puberty. These mammalian ovarian glycolipids are complementary also to the anti-A/Tn cross-reactive Helix pomatia agglutinin (HPA), a molluscan defense protein, emerging from the coat proteins of fertilized eggs and reflecting the snail-intrinsic, reversible O-GalNAc glycosylations. The hexameric structure of this primitive invertebrate defense protein gives rise to speculation regarding an evolutionary relationship to the mammalian nonimmune, anti-A-reactive immunoglobulin M (IgM) molecule. Hypothetically, this molecule obtains its complementarity from the first step of protein glycosylations, initiated by GalNAc via reversible O-linkages to peptides displaying Ser/Thr motifs, whereas the subsequent transferase depletion completes germ cell maturation and cell renewal, associated with loss of glycosidic bonds and release of O-glycan-depleted proteins, such as complementary IgM revealing the structure of the volatilely expressed "lost" glycan carrier through germline Ser residues. Consequently, the evolutionary/developmental first glycosylations of proteins appear metabolically related or identical to that of the mucin-type, potentially "aberrant" monosaccharide GalNAcα1-O-Ser/Thr-R, also referred to as the Tn (T "nouvelle") antigen, and explain the anti-Tn cross-reactivity of human innate or "natural" anti-A-specific isoagglutinin and the pronounced occurrence of cross-reactive anti-Tn antibody in plasma from humans with histo-blood group O. In fact, A-allelic, phenotype-specific GalNAc glycosylation of plasma proteins does not occur in human blood group O, affecting anti-Tn antibody levels, which may function as a growth regulator that contributes to a potential survival advantage of this group in the overall risk of developing cancer when compared with non-O blood groups.

Dataset Information

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

Publications

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

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