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

Thomas J M Michiels,Gideon F A Kersten,Hugo D Meiring,Wim Jiskoot,Bernard Metz

doi:10.1038/s41598-020-68248-z

Thomas J M Michiels, Gideon F A Kersten + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-68248-z

Copy DOI

Abstract

Enzymatic degradation of protein antigens by endo-lysosomal proteases in antigen-presenting cells is crucial for achieving cellular immunity. Structural changes caused by vaccine production process steps, such as formaldehyde inactivation, could affect the sensitivity of the antigen to lysosomal proteases. The aim of this study was to assess the effect of the formaldehyde detoxification process on the enzymatic proteolysis of antigens by studying model proteins. Bovine serum albumin, β-lactoglobulin A and cytochrome c were treated with various concentrations of isotopically labelled formaldehyde and glycine, and subjected to proteolytic digestion by cathepsin S, an important endo-lysosomal endoprotease. Degradation products were analysed by mass spectrometry and size exclusion chromatography. The most abundant modification sites were identified by their characteristic MS doublets. Unexpectedly, all studied proteins showed faster proteolytic degradation upon treatment with higher formaldehyde concentrations. This effect was observed both in the absence and presence of glycine, an often-used excipient during inactivation to prevent intermolecular crosslinking. Overall, subjecting proteins to formaldehyde or formaldehyde/glycine treatment results in changes in proteolysis rates, leading to an enhanced degradation speed. This accelerated degradation could have consequences for the immunogenicity and the efficacy of vaccine products containing formaldehyde-inactivated antigens.

Highlights

Enzymatic degradation of protein antigens by endo-lysosomal proteases in antigen-presenting cells is crucial for achieving cellular immunity
Enzymatic degradation of antigens is a crucial step in the process of acquiring cellular immunity, e.g., through the induction of antigen specific T-helper cells or cytotoxic T-cells
That study used trypsin as an enzyme, which cleaves after lysine and arginine residues. Both of these amino acids are susceptible to formaldehyde modification, especially the latter

Summary

Free N termini

Being converted in substantial amounts.[15]. Formaldehyde modification of trypsin’s cleavage sites is very likely to affect the substrate’s degradation kinetics, as lysine modifications inhibit digestion by trypsin, but this might be less relevant for lysosomal degradation in antigen-presenting cells (APCs).[18]. To assess the effect of formaldehyde treatment on enzymatic protein processing three formaldehyde-treated model proteins were prepared: bovine serum albumin (BSA), β-lactoglobulin A and cytochrome c. These proteins were chosen for their structural diversity, commercial availability and their low toxicity (as compared to toxins). These proteins were treated with increasing concentrations of formaldehyde and glycine. They were subjected to proteolytic digestion by cathepsin S, an important endo-lysosomal enzyme.[19] The degradation of the intact proteins was monitored by size exclusion chromatography (SEC) and the resulting peptides were identified and quantified using nanoscale liquid chromatography-mass spectrometry (LC–MS)

Results and discussion

Author contributions

Additional information