Hypoxia-induced post-translational changes in red blood cell protein map of newborns.
Tyrosine (Tyr) phosphorylation is implicated in the modification of several erythrocyte functions, such as metabolic pathways and membrane transport, as well as in signal transduction systems. Here we describe the map of Tyr-phosphorylated soluble proteins of newborn red blood cells (RBC) using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. We tested the hypothesis that a hypoxic environment and subsequent reoxygenation promote post-translational changes in the RBC protein map of newborns, in addition to desferrioxamine (DFO)-chelatable iron (DCI) release and methemoglobin (MetHb) formation. Umbilical cord blood RBC were incubated under hypoxic conditions for 16 h at 37 degrees C, and subsequently for 8 h under aerobic conditions. Control erythrocytes were incubated under aerobic conditions at 37 degrees C for the period of the experiment, i.e. for 24 h. Tyr-phosphorylation proteins were assessed using advanced high-resolution two-dimensional electrophoresis, 2-D immunoblot analysis with anti-phosphotyrosine (anti-pTyr) antibodies, and computer-aided electrophoretogram analysis. Higher DCI release and MetHb formation were observed in newborn RBC incubated under hypoxic conditions than in those incubated aerobically. Different immunoreactivity patterns with anti-pTyr antibodies were also observed between newborn RBC incubated under hypoxic conditions and controls. A hypoxic environment is a factor promoting DCI release, a well-known condition of oxidative stress. This is the first map of Tyr-phosphorylated soluble proteins of newborn RBC obtained using an in vitro model simulating RBC reoxygenation at birth after an intrauterine hypoxic event. Our results suggest that hypoxia increases Tyr-phosphorylation of antioxidant proteins, protecting RBC against oxidative stress.
Marzocchi
Barbara
B
Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Italy.
Ciccoli
Lucia
L
Tani
Chiara
C
Leoncini
Silvia
S
Rossi
Viviana
V
Bini
Luca
L
Perrone
Serafina
S
Buonocore
Giuseppe
G
D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting; D066298:In Vitro Techniques; D007231:Infant, Newborn; D008706:Methemoglobin / Q000737:chemistry; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D016622:Silver Staining; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D014471:Umbilical Veins / Q000166:cytology
D019000:Phosphotyrosine; D014443:Tyrosine; D008706:Methemoglobin; D010100:Oxygen