Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing

Muts, Remy M.; den Boer, Maurits A.; Bardoel, Bart W.; Aerts, Piet C.; de Haas, Carla J. C.; Heck, Albert J. R.; Rooijakkers, Suzan H. M.; Heesterbeek, Dani A. C.

doi:10.1038/s41598-023-46026-x

Cited by 2 publications

(1 citation statement)

References 65 publications

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“…Production of IgM Constructs. Recombinant anti-StrepTag IgM with or without J-chain were produced as extensively described by Muts et al 36 In short, the variable heavy and light chains of anti-StrepTagII IgG (WO 2015/ 067768 A1, 2015) with upstream KOZAK and HAVT20 signal peptide were cloned into adapted pcDNA34 vectors (Thermo-Fisher Scientific), upstream the IgM heavy and kappa light chain constant regions. 37 A plasmid coding for J-chain expression was kindly provided by Theo Rispens.…”

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confidence: 99%

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

Yin,

Deslignière,

Mokiem

et al. 2024

J. Am. Soc. Mass Spectrom.

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Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab') 2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab') 2 subunits is much slower than the other four F(ab') 2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

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Section: ) (Red Dashed Line) (B)mentioning

confidence: 99%

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

Yin,

Deslignière,

Mokiem

et al. 2024

J. Am. Soc. Mass Spectrom.

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Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization

Masson,

Káradóttir,

Lans

et al. 2024

Preprint

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The Gram-negative bacterium Klebsiella pneumoniae is an important human pathogen. Its treatment has been complicated by the emergence of multi-drug resistant strains. The human complement system is an important part of our innate immune response that can directly kill Gram-negative bacteria by assembling membrane attack complex (MAC) pores into the bacterial outer membrane. To resist this attack, Gram-negative bacteria can modify their lipopolysaccharide (LPS). Especially the decoration of the LPS outer core with the O-antigen polysaccharide has been linked to increased bacterial survival in serum, but not studied in detail. In this study, we characterized various clinical Klebsiella pneumoniae isolates and show that expression of the LPS O1-antigen correlates with resistance to complement-mediated killing. Mechanistic data reveal that the O1-antigen does not inhibit C3b deposition and C5 conversion. In contrast, we see more efficient formation of C5a, and deposition of C6 and C9 when an O-antigen is present. Further downstream analyses revealed that the O1-antigen prevents correct insertion and polymerization of the final MAC component C9 into the bacterial membrane. Altogether, we show that the LPS O1-antigen is a key determining factor for complement resistance by K. pneumoniae and provide insights into the molecular basis of O1-mediated MAC evasion.

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Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing

Cited by 2 publications

References 65 publications

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS

Klebsiella LPS O1-antigen prevents complement-mediated killing by inhibiting C9 polymerization

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