PXD057808-1

PXD057808 is an original dataset announced via ProteomeXchange.

Title	Small angle X-ray scattering of engineered antigen-binding fragments: the case of glycosylated Fab from the Mannitou IgM antibody
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-12-30
AnnouncementXML	Submission_2024-12-30_01:28:53.933.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	BRAY FABRICE
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; deaminated residue; complex glycosylation; iodoacetamide derivatized residue
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2024-11-12 12:09:56	ID requested
⏵ 1	2024-12-30 01:28:54	announced

10.1107/s2053230x24012159;

Semwal S, Karamolegkou M, Flament S, Raouraoua N, Verstraete K, Thureau A, Wien F, Bray F, Savvides SN, Bouckaert J, Small-angle X-ray scattering of engineered antigen-binding fragments: the case of glycosylated Fab from the Mannitou IgM antibody. Acta Crystallogr F Struct Biol Commun, 81(Pt 1):19-29(2025) [pubmed]

submitter keyword: IgM, Glycosylation,Maniitou

fabrice bray
contact affiliation	MSAP UAR 3290 Bat C4 avenue Paul Langevin 59655 Villeneuve d'ascq
contact email	fabrice.bray@univ-lille.fr
lab head
BRAY FABRICE
contact affiliation	MSAP USR 3290
contact email	fabrice.bray@univ-lille.fr
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD057808
     1. Label: PRIDE project
     2. Name: Small angle X-ray scattering of engineered antigen-binding fragments: the case of glycosylated Fab from the Mannitou IgM antibody