Identification of Formaldehyde-Induced Modifications in Diphtheria Toxin

Abstract

Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH 3 , (2) formaldehyde, (3) formaldehyde and NaCNBH 3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatographyeelectrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD þ-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD þ-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4 þ T-cell epitopes were affected to some extent, one universal CD4 þ Tcell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine.

Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

“…Functionally the process affected to some extension CD4þ T-cell epitopes but universal CD4þ Tcell epitope remained almost completely unaltered [58]. In our study, we can conclude that apparently the detoxification process does not interfere in the recognition of linear B epitopes, since antibodies present in the sera of mice immunized recognized almost of all linear epitopes of the DTx.…”

“…The current production process for DTx vaccines is largely based on the traditional methods first used to detoxify DTx [57]. Recently Metzel et al, [58] reveled the chemical modification of the DTx induced by formaldehyde treatment. The detoxification used for vaccine preparation typically resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments.…”

Formaldehyde treatment of proteins enhances proteolytic degradation by the endo-lysosomal protease cathepsin S

“…The challenging neutral losses from CID fragmentation can often be avoided by using Electron Transfer Dissociation (ETD) fragmentation, but this requires multiply charged (z > 2) species at relatively high abundance. 21 To overcome these limitations and avoid bias, only the formation kinetics of some of the most abundant peptides that are considered inert, i.e. , not modifiable by formaldehyde, are plotted in Fig.…”

“…The isotopically labelled digestion samples CYT-F128-1 and CYT-FG128-1 after 48 h of digestion were analysed with MsXelerator (MsMetrix) to obtain a list of isotopic doublets of peptides that should contain formaldehyde or formaldehyde-glycine adducts. 21 The 48 h samples were then measured again with a targeted analysis. Subsequently, another PEAKS search was performed to identify these modified products.…”

A streamlined mass spectrometry-based proteomics workflow for large scale FFPE tissue analysis

“…Together, these data demonstrate efficient protein extraction using our organic solvent-based workflow, including chromatin bound proteins. Formaldehyde crosslinking of FFPE tissue preserves tissue architecture, predominantly via stable methylene bridges between basic amino acid residues [23,24]. High proteome coverage of FFPE tissue therefore requires efficient formaldehyde de-crosslinking to reverse unwanted and variable chemical modifications that may obscure peptide identification.…”