Abstract
Communication through cell surface receptors is crucial for maintaining immune homeostasis, coordinating the immune response and pathogen clearance. This is dependent on the interaction of cell surface receptors with their ligands and requires functionally active conformational states. Thus, immune cell function can be controlled by modulating the structure of either the receptor or the ligand. Reductive cleavage of labile disulfide bonds can mediate such an allosteric change, resulting in modulation of the immune system by a hitherto little studied mechanism. Identifying proteins with labile disulfide bonds and determining the extent of reduction is crucial in elucidating the functional result of reduction. We describe a mass spectrometry-based method—thiol identification and quantitation (SH-IQ)—to identify, quantify and monitor such reduction of labile disulfide bonds in primary cells during immune activation. These results provide the first insight into the extent and dynamics of labile disulfide bond reduction in leucocyte cell surface proteins upon immune activation. We show that this process is thiol oxidoreductase-dependent and mainly affects activatory (e.g. CD132, SLAMF1) and adhesion (CD44, ICAM1) molecules, suggesting a mechanism to prevent over-activation of the immune system and excessive accumulation of leucocytes at sites of inflammation.
Highlights
The adaptive immune system provides the host with a powerful defence against invading pathogens and diseases by mounting antibody or cellular responses towards pathogens and infected or malignant cells [1]
We have previously developed a first-generation, non-quantitative mass spectrometry-based method that identified a vast number of leucocyte cell surface proteins with supposed redox labile disulfide bonds [14]
To develop a quantitative mass spectrometry-based method to analyse the reductive cleavage of labile disulfide bonds
Summary
The adaptive immune system provides the host with a powerful defence against invading pathogens and diseases by mounting antibody or cellular responses towards pathogens and infected or malignant cells [1]. It is becoming increasingly clear that some disulfide bonds can be post-translationally cleaved in by thiol oxidoreductase enzymes [6] These labile disulfide bonds are either catalytic (found at the active site of cysteine thiol reductase and thiol isomerase enzymes) or allosteric (found in protein motifs that when reduced mediate an allosteric change in the protein structure modulating protein function). We have previously developed a first-generation, non-quantitative mass spectrometry-based method that identified a vast number of leucocyte cell surface proteins with supposed redox labile disulfide bonds [14]. We report the development of SH-IQ, a label-free quantitative proteomics workflow for the identification and quantitation of labile disulfide bond reduction This showed that only a subset of the 86 proteins previously identified to contain labile disulfide bonds [14] are significantly reduced using the chemical reductant TCEP. SH-IQ revealed the dynamic and highly regulated reduction of labile disulfide bonds during immune activation in a mixed lymphocyte reaction (MLR)
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