Title :

Small-angle X-ray scattering of engineered antigen-binding fragments: the case of glycosylated Fab from the Mannitou IgM antibody

Author(s) :

Semwal, Shubham [Auteur]
Karamolegkou, Maria [Auteur]
Flament, Stéphanie [Auteur]
Raouraoua, Nessim [Auteur]
Verstraete, Kenneth [Auteur]
Thureau, Aurélien [Auteur]
Wien, Frank [Auteur]
Bray, Fabrice [Auteur]
Savvides, Savvas N. [Auteur]
Bouckaert, Julie [Auteur]
unité mixte CNRS

Journal title :

Acta Crystallographica Section F Structural Biology Communications

Volume number :

81

Pages :

19-29

Publisher :

International Union of Crystallography (IUCr)

Publication date :

2025-01-01

ISSN :

2053-230X

HAL domain(s) :

Sciences du Vivant [q-bio]

English abstract : [en]

Monoclonal antibodies recognizing nonprotein antigens remain largely underrepresented in our understanding of the molecular repertoire of innate and adaptive immunity. One such antibody is Mannitou, a murine IgM that ...
Show more >Monoclonal antibodies recognizing nonprotein antigens remain largely underrepresented in our understanding of the molecular repertoire of innate and adaptive immunity. One such antibody is Mannitou, a murine IgM that recognizes paucimannosidic glycans. In this work, we report the production and purification of the recombinant antigen-binding fragment (Fab) of Mannitou IgM (Mannitou Fab) and employ a combination of biochemical and biophysical approaches to obtain its initial structural characterization. To this end, recombinant Mannitou Fab comprising the light chain (VL-CL) and heavy chain (VH-Cμ1) was produced in HEK293 FreeStyle cells and purified by cobalt-affinity chromatography followed by size-exclusion chromatography (SEC), which revealed two distinct oligomeric states consistent with a predominant monomeric form and a minor dimeric form. We employed SEC inline with multi-angle light scattering (SEC-MALS) and SEC coupled to small-angle X-ray scattering (SEC-SAXS) to establish that Mannitou Fab indeed adopts monomeric and dimeric forms in solution. Interestingly, Mannitou Fab is N-glycosylated at Asn164 of the heavy chain via HexNAc(5)Hex(6)Fuc(1–3) as revealed by mass spectrometry. We leveraged this information in conjunction with predicted structures of Mannitou Fab to facilitate the interpretation and modelling of SAXS data, leading to a plausible model for glycosylated Mannitou Fab. Analysis of the two chromatographically isolatable forms of Mannitou Fab using synchrotron-radiation circular dichroism revealed that the heat-denaturated Mannitou Fab monomer shares similar secondary-structural elements with the Mannitou Fab dimer, indicating that the latter may be misfolded. Collectively, the findings of this study will set the stage for future structural studies of Mannitou Fab and contribute to our understanding of possible side products due to misfolding during the production of recombinant Fabs, highlighting the importance of glycosylation in obtaining stable and monodisperse monomeric forms of recombinant Fabs.Show less >

Language :

Anglais

Audience :

Internationale

Popular science :

Non

Administrative institution(s) :

Université de Lille
CNRS

Collections :

• Miniaturisation pour la Synthèse, l'Analyse et la Protéomique (MSAP) - USR 3290
• Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576

Research team(s) :

Computational Molecular Systems Biology

Submission date :

2025-01-20T10:49:55Z