Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

Giuntini, Stefano; Balducci, Evita; Cerofolini, Linda; Ravera, Enrico; Fragai, Marco; Luchinat, Claudio

doi:10.1002/ange.201709274

Cited by 4 publications

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References 52 publications

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“…The conjugation positions can be identified by mass spectrometry, but it is still a challenge to map conjugation sites in populations of polydisperse molecules. Some reports on capsular polysaccharides (CPS) show that solid-state NMR spectroscopy is a working alternative to determine PS/protein conjugation pattern and the degree of conjugation; however, it has not yet been reported for Salmonella Typhi, iNTS or Shigella conjugated vaccines (Giuntini et al 2017 ). To determine sugar:protein ratio, colorimetric tests are still popular due to their low cost and relative simplicity, for example anthrone and a colorimetric protein concentration assay (Leyva et al 2008 ; Ravenscroft et al 2015b ).…”

Section: Part IImentioning

confidence: 99%

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

et al. 2021

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The global spread of enteric disease, the increasingly limited options for antimicrobial treatment and the need for effective eradication programs have resulted in an increased demand for glycoconjugate enteric vaccines, made with carbohydrate-based membrane components of the pathogen, and their precise characterisation. A set of physico-chemical and immunological tests are employed for complete vaccine characterisation and to ensure their consistency, potency, safety and stability, following the relevant World Health Organization and Pharmacopoeia guidelines. Variable requirements for analytical methods are linked to conjugate structure, carrier protein nature and size and O-acetyl content of polysaccharide. We investigated a key stability-indicating method which measures the percent free saccharide of Salmonella enterica subspecies enterica serovar Typhi capsular polysaccharide, by detergent precipitation, depolymerisation and HPAEC-PAD quantitation. Together with modern computational approaches, a more precise design of glycoconjugates is possible, allowing for improvements in solubility, structural conformation and stability, and immunogenicity of antigens, which may be applicable to a broad spectrum of vaccines. More validation experiments are required to establish the most effective and suitable methods for glycoconjugate analysis to bring uniformity to the existing protocols, although the need for product-specific approaches will apply, especially for the more complex vaccines. An overview of current and emerging analytical approaches for the characterisation of vaccines against Salmonella Typhi and Shigella species is described in this paper. This study should aid the development and licensing of new glycoconjugate vaccines aimed at the prevention of enteric diseases.

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Section: Part IImentioning

confidence: 99%

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

et al. 2021

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Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside

Marra

Dong

et al. 2018

Chemistry A European J

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Protein glycosylation is the most complex post‐translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l‐asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact‐Ar‐OSO2F) with the ϵ‐NH2 group of lysine residues of the proteins. This metal‐free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ‐NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact‐Ar‐OSO2F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact‐Ar‐OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2‐NH) linkage.

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Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

Cerofolini

Giuntini

Carlon

et al. 2019

Chemistry A European J

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Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to “evade” the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X‐ray crystallography.

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Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

Cited by 4 publications

References 52 publications

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

Glycoconjugate vaccines against Salmonella enterica serovars and Shigella species: existing and emerging methods for their analysis

Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics

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