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Protein phosphorylation of neutrophils from normal children and patients with chronic granulomatous disease
Insights
Protein phosphorylation in neutrophils was studied in healthy children and those with chronic granulomatous disease (CGD). Specific protein changes linked to superoxide production activation were identified and found unaffected in CGD neutrophils.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophils play a critical role in the innate immune system, particularly in combating microbial infections through the production of reactive oxygen species.
- Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by a defect in the respiratory burst, leading to recurrent infections.
- Understanding the molecular mechanisms regulating neutrophil activation, specifically protein phosphorylation, is crucial for elucidating immune responses and disease pathogenesis.
Purpose of the Study:
- To investigate the patterns of protein phosphorylation and dephosphorylation in neutrophils from normal children and CGD patients.
- To identify specific proteins involved in the activation of superoxide production in neutrophils.
- To determine if the observed protein phosphorylation changes are altered in neutrophils from CGD patients.
Main Methods:
- Two-dimensional gel electrophoresis and autoradiography were employed to analyze protein phosphorylation.
- Densitometric scanning was used to quantify changes in radioactivity of protein spots.
- Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), and NaF to induce activation.
- Inhibitors of superoxide production (2-deoxyglucose, W-7) and non-inhibitors (dibutyryl cAMP, KCN, EGTA) were used to probe signaling pathways.
Main Results:
- Stimulation of normal neutrophils with PMA, FMLP, and NaF altered the radioactivity of 11 protein spots.
- Phosphorylation of two specific proteins (Mr = 48,000 and 62,000) was inhibited by agents that also inhibit superoxide production.
- These specific phosphorylation changes were not affected by agents that do not inhibit superoxide production.
- Neutrophils from CGD patients exhibited similar protein phosphorylation changes upon stimulation compared to normal neutrophils.
Conclusions:
- The proteins with Mr = 48,000 and 62,000 are likely involved in the activation process of superoxide production in neutrophils.
- The (de)phosphorylation of these key proteins appears to be independent of the defect causing chronic granulomatous disease.
- These findings suggest that the regulatory mechanisms of protein phosphorylation related to superoxide production activation are intact in CGD neutrophils, despite the functional deficiency in respiratory burst.
Abstract:
The phosphorylation and dephosphorylation of proteins in neutrophils from normal children and patients with chronic granulomatous disease (CGD) were studied with two-dimensional gel electrophoresis and autoradiography, followed by densitometric scanning. In normal neutrophils the radioactivities of 11 spots among approximately 50 radioactive spots were changed by stimulation with phorbol 12-myristate 13-acetate (PMA) and 6 of the 11 spots were also changed by stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and NaF. The phosphorylation of only two spots (Mr = 48 000 and 62 000) was inhibited by 2-deoxyglucose and N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), which inhibits superoxide production, while it was not affected by dibutyryl cAMP, KCN and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), which do not affect superoxide production. The observation indicates that the Mr = 48 000 and 62 000 proteins may be involved in the activation process of superoxide production. When the neutrophils of four male and two female CGD patients were examined, the changes in 11 spots on stimulation were similar to those of normal children, indicating that the (de)phosphorylation of the proteins which seems to be involved in the activation process is not affected in CGD neutrophils.