Protein tyrosine nitration of aldolase in mast cells: a plausible pathway in nitric oxide-mediated regulation of mast cell function.
NO is a short-lived free radical that plays a critical role in the regulation of cellular signaling. Mast cell (MC)-derived NO and exogenous NO regulate MC activities, including the inhibition of MC degranulation. At a molecular level, NO acts to modify protein structure and function through several mechanisms, including protein tyrosine nitration. To begin to elucidate the molecular mechanisms underlying the effects of NO in MCs, we investigated protein tyrosine nitration in human MC lines HMC-1 and LAD2 treated with the NO donor S-nitrosoglutathione. Using two-dimensional gel Western blot analysis with an anti-nitrotyrosine Ab, together with mass spectrometry, we identified aldolase A, an enzyme of the glycolytic pathway, as a target for tyrosine nitration in MCs. The nitration of aldolase A was associated with a reduction in the maximum velocity of aldolase in HMC-1 and LAD2. Nuclear magnetic resonance analysis showed that despite these changes in the activity of a critical enzyme in glycolysis, there was no significant change in total cellular ATP content, although the AMP/ATP ratio was altered. Elevated levels of lactate and pyruvate suggested that S-nitrosoglutathione treatment enhanced glycolysis. Reduced aldolase activity was associated with increased intracellular levels of its substrate, fructose 1,6-bisphosphate. Interestingly, fructose 1,6-bisphosphate inhibited IgE-mediated MC degranulation in LAD2 cells. Thus, for the first time we report evidence of protein tyrosine nitration in human MC lines and identify aldolase A as a prominent target. This posttranslational nitration of aldolase A may be an important pathway that regulates MC phenotype and function.
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
Yokananth
Y
Sekar
Tae Chul
TC
Moon
Carolyn M
CM
Slupsky
A Dean
AD
Befus
D015550:Cell Degranulation / Q000276:immunology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006019:Glycolysis / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D008407:Mast Cells / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000276:immunology; D026422:S-Nitrosoglutathione / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D013379:Substrate Specificity / Q000276:immunology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*
D020030:Nitric Oxide Donors; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D026422:S-Nitrosoglutathione; D005634:Fructose-Bisphosphate Aldolase