Nitration of surfactant protein A results in decreased ability to aggregate lipids.
We assessed the extent to which nitration of surfactant protein (SP) A, isolated from the bronchoalveolar lavage of patients with alveolar proteinosis, alters its ability to enhance lipid aggregation, bind lipids, and act synergistically with surfactant apoproteins B and C (SP-B, SP-C) in lowering the surface activity of surfactant lipids. SP-A was treated with various concentrations of tetranitromethane (TNM) at pH 6, 7.4, 8, or 10. Depending on the pH, TNM acts either as a nitrating (pH > or = 7.4) or an oxidizing agent (pH < or = 6). Exposure of SP-A to TNM (0.1-1 mM) at pH 7.4 or 8 for 30 min resulted in dose-and pH-dependent increases in nitrotyrosine, detected by Western blotting, enzyme-linked immunosorbent assay, and direct amino acid analysis. Treatment of SP-A with 0.5 mM TNM decreased its ability to aggregate lipids by 30% at pH 7.4, and 90% at pH 8, but had no effect on the disulfide-dependent oligomeric state of SP-A. In contrast, SP-A exposed to 1 mM TNM at pH 6 had background levels of nitrotyrosine and exhibited normal lipid aggregation properties. TNM, but not a hydroxyl radical-generating system, resulted in a pH-dependent loss of SP-A fluorescence, suggesting that tryptophan also may have been nitrated. Nitration of SP-A did not affect its ability to bind lipids. In addition, SP-A (1-3% by weight), treated with 0.25-0.5 mM TNM at pH 8, restored the surface-active properties of calf lung surfactant extract, previously damaged by exposure to peroxynitrite. We conclude that tyrosine nitration selectively inhibits the SP-A-mediated lipid aggregation without affecting its ability to bind lipids.
Haddad
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Department of Pediatrics, University of Alabama at Birmingham 35233-6810, C, USA.
Zhu
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Ischiropoulos
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Matalon
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D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000502:physiology*; D009566:Nitrates / Q000378:metabolism*; D011510:Proteolipids / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013500:Surface Tension / Q000187:drug effects; D013774:Tetranitromethane / Q000494:pharmacology; D014364:Tryptophan / Q000494:pharmacology
D000596:Amino Acids; D008055:Lipids; D009566:Nitrates; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D013438:Sulfhydryl Compounds; D014364:Tryptophan; D013774:Tetranitromethane