Abstract
Sickle erythrocytes are known to undergo excessive auto-oxidation, resulting in the generation of increased intracellular levels of several species of free radical oxidants. This environment is likely to enhance the accumulation of oxidative lesions by membrane components, although, as yet, this has been shown directly only for the sickle membrane phospholipids. We examined the oxidative status of protein 4.1, a major component of the human erythrocyte protein skeleton. We found that protein 4.1 isolated from sickle erythrocytes bound approximately 4-fold less to protein 4.1-stripped membranes than did the normal protein. The binding defect was inherent in the sickle protein and not in its membrane-binding site(s) since normal protein 4.1 bound to sickle protein 4.1-stripped inside-out vesicles similar to normal protein 4.1-stripped inside-out vesicles. Sickle membranes, in particular spectrin-depleted inside-out vesicles, contained less protein 4.1 than normal membranes. Purified sickle protein 4.1 contained 20-40% high molecular weight aggregated protein (Mr greater than 200,000), whereas the purified normal protein contained approximately 10% high molecular weight protein. The high molecular weight protein was immunoreactive with antibodies to protein 4.1 but not with antibodies to spectrin, ankyrin, band 3, glycophorin, or hemoglobin, suggesting that the high molecular weight protein was cross-linked protein 4.1 and not a complex of protein 4.1 and some other membrane protein(s). Purified sickle protein 4.1 was eluted from an anion-exchange resin at a higher salt concentration than normal protein 4.1. Oxidizing normal protein 4.1 with diamide resulted in an anion-exchange elution pattern similar to the sickle protein, suggesting that oxidation can affect protein surface charge. Activated thiol beads bound one-half as much sickle protein 4.1 as normal protein 4.1 when both were solubilized directly from membranes, demonstrating that thiol oxidation had occurred in vivo. Direct quantification of protein thiols revealed that the sickle protein contained 1-2 mol% fewer cysteines/protein 4.1 monomer than did the normal protein. By amino acid analysis, sickle protein 4.1 was found to contain less methionine and tyrosine than did the normal protein and contained approximately 1 mol% cysteic acid, whereas the normal protein did not contain any cysteic acid. Taken together, our results strongly suggest that sickle protein 4.1 has sustained oxidative damage in vivo. This damage can alter the functional properties of the sickle protein and may be an underlying factor in the myriad of membrane abnormalities reported in sickle erythrocytes.
Highlights
Sickle erythrocytesare known to undergo excessive Takentogether,ourresultsstrongly suggest that auto-oxidation, resulting in the generatioofnincreased sickle protein 4.1 has sustained oxidative damage in intracellular levels of several species of free radical vivo. This damage can alter the functional properties oxidants. This environment is likely to enhance the of the sickle protein andmay be an underlying factor accumulation of oxidative lesions by membrane com- in the myriad of membrane abnormalities reported in ponents, as yet, this hasbeen shown directly sickle erythrocytes. only for the sicklemembrane phospholipids
We examined the oxidative status of protein 4.1, a major component of the human erythrocyte protein skeleton
Recent evidence by Platt et al [31] furthersuggests that 4.1 compared to normal protein4.1 cannot, by itself, explain acquireddamageto specific components of the sicklecell the 4-fold decrease in membrane binding using the purified cytoskeleton adversely affects some aspects of protein func- sickle protein and protei4n.1-stripped membranes
Summary
From the Children’s Hospital Oakland, ResearchInstitute, Oakland, California 94609. Sickle erythrocytesare known to undergo excessive Takentogether,ourresultsstrongly suggest that auto-oxidation, resulting in the generatioofnincreased sickle protein 4.1 has sustained oxidative damage in intracellular levels of several species of free radical vivo. Determined by subjecting 20-pg samples to NaDodS0,-PAGE This procedure resulted in a acrylamide) under reducing conditions using the discontinuous buffer concentration of DTT in the dialyzed protein of CO. p ~ R.emoval system of Laemmli [16]. Rebinding assays were performed in a total volume of0.2 ml, and a large excess of gel buffer to remove the DTTimmediately prior to HSVs were collected by centrifugation (40,000 X g) for 30 min a t 4 "C mixing with the gel since DTT interferes with protein-gel binding. The following which time the columns were drained and the filtrate was washed pellets were solubilized in 1.5% NaDodSO,, 5 mM DTT and collected (unboundprotein), dialyzed against 5P8 for 2h using analyzed by NaDodS0,-PAGE (9% acrylamide) under reducing con- Spectrapor-2 dialysis membrane, lyophilized, redissolved in 0.1 ml of ditions [16]. The hydrolyzed protein was lyophilizedand redissolvedin 0.2 M sodium citrate buffer, pH 2.2, and total amino acid composition was analyzed on a Dionex amino acid analyzer
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