Dataset Information

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

ABSTRACT: The antigen binding fragment (Fab) of Mannitou IgM was recombinantly produced in HEK293 FreeStyle cells and purified over a cobalt-affinity chromatography followed by size exclusion chromatography (SEC). Besides the presence of the monomeric Mannitou Fab assembly, existing of the light chain (VL-CL) and heavy chain (VH-Cμ1), higher order oligomers have also been observed. In this work, we critically analysed the content of the monomeric Fab and of a multimer with the molecular mass of a dimer, using SEC-MALS, SEC-SAXS, mass spectrometry and synchrotron radiation circular dichroism (SRCD). Monomeric Mannitou Fab was found to be glycosylated on Asn168 of the heavy chain, with an N-linked complex glycan with the composition HexNAc(5)Hex(6)Fuc(2). Addition of the glycan to the AlphaFold3 model, using GlycoSHIELD, significantly improved the fitting to the SAXS data and the best fitting glycan conformation was revealed. Glycosylation improved the stability of the Fab model and abolished fitting towards multi-state models of the Fab monomer. SRCD indicated that when the Mannitou Fab monomer heat denatured, it obtained secondary structure elements close to the multimer with the molecular mass of a dimer. A credible model to fit the SAXS of the dimer could not be found despite a search using MassiveFold, an offspring of AlphaFold2. The findings from this study contribute to the understanding of the occurrence of Fab side products through misfolding and the possibility of domain swapping in the engineering and design of recombinant Fabs. They highlight the importance of glycosylation of Mannitou Fab to maintain a stable monomeric form and to prevent unwanted interactions, thus improving its potential as a therapeutic candidate.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: BRAY FABRICE

LAB HEAD: fabrice bray

PROVIDER: PXD057808 | Pride | 2024-12-30

REPOSITORIES: Pride

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

Semwal Shubham S Karamolegkou Maria M Flament Stéphanie S Raouraoua Nessim N Verstraete Kenneth K Thureau Aurélien A Wien Frank F Bray Fabrice F Savvides Savvas N SN Bouckaert Julie J

Acta crystallographica. Section F, Structural biology communications 20250101 Pt 1

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 characteri ...[more]

PMID: 39718025

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Project description:The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

			Action	DRS
	P18-1.raw	Raw
	P18-2.raw	Raw
	P18-Fab.csv	Csv
	P18-HFab.csv	Csv
	checksum.txt	Txt