Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β2on Monocytes via NADPH Oxidase.
Physiological stimuli such as thrombin, or pathological stimuli such as lysophosphatidic acid (LPA), activate platelets. The activated platelets bind to monocytes through P-selectin-PSGL-1 interactions but also release the contents of their granules, commonly called "platelet releasate". It is known that monocytes in contact with platelet releasate produce reactive oxygen species (ROS). Reversible cysteine oxidation by ROS is considered to be a potential regulator of protein function. In a previous study, we used THP-1 monocytic cells exposed to LPA- or thrombin-induced platelet releasate and a modified biotin switch assay to unravel the biological processes that are influenced by reversible cysteine oxidation. To gain a better understanding of the redox regulation of monocytes in atherosclerosis, we have now altered the modified biotin switch to selectively quantify protein sulfenic acid, a subpopulation of reversible cysteine oxidation. Using arsenite as reducing agent in the modified biotin switch assay, we were able to quantify 1161 proteins, in which more than 100 sulfenic acid sites were identified. Bioinformatics analysis of the quantified sulfenic acid sites highlighted the relevant, previously missed biological process of monocyte transendothelial migration, which included integrin β2. Flow cytometry validated the activation of LFA-1 (αLβ2) and Mac-1 (αMβ2), two subfamilies of integrin β2complexes, on human primary monocytes following platelet releasate treatment. The activation of LFA-1 was mediated by ROS from NADPH oxidase (NOX) activation. Production of ROS and activation of LFA-1 in human primary monocytes were independent of P-selectin-PSGL-1 interaction. Our results proved the modified biotin switch assay to be a powerful tool with the ability to reveal new regulatory mechanisms and identify new therapeutic targets.
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Ru
R
Li
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Cordula
C
Klockenbusch
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Liwen
L
Lin
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Honghui
H
Jiang
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Shujun
S
Lin
The Biomedical Research Centre, ‡Department of Chemistry, and §Centre for Blood Research, University of British Columbia , Vancouver, Canada V6T 1Z3.
Juergen
J
Kast
D018053:Arsenites; D001792:Blood Platelets / Q000378:metabolism*; D018821:CD18 Antigens / Q000378:metabolism*; D019295:Computational Biology; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism; D022142:Secretory Vesicles; D013434:Sulfenic Acids / Q000032:analysis*; D057705:Transendothelial and Transepithelial Migration
D018053:Arsenites; D018821:CD18 Antigens; D016169:Lymphocyte Function-Associated Antigen-1; D016177:Macrophage-1 Antigen; D011506:Proteins; D017382:Reactive Oxygen Species; D013434:Sulfenic Acids; D019255:NADPH Oxidases; C015001:arsenite
THP-1 cells; biotin switch assay; human primary monocyte; integrin β2; protein sulfenic acid