Leavey Patrick J, Gonzalez-Aller Carolina, Thurman Gail, Kleinberg Michael, Rinckel Lori, Ambruso Daniel W, Freeman Stefanie, Kuypers Frans A, Ambruso Daniel R
Bonfils Blood Center, Denver, Colorado 80230, USA.
J Biol Chem. 2002 Nov 22;277(47):45181-7. doi: 10.1074/jbc.M202869200. Epub 2002 Jul 16.
Production of toxic oxygen metabolites provides a mechanism for microbicidal activity of the neutrophil. The NADPH oxidase enzyme system initiates the production of oxygen metabolites by reducing oxygen to form superoxide anion (O(2)()). With stimulation of the respiratory burst, cytosolic oxidase components, p47(phox), p67(phox), and Rac, translocate to the phagolysomal and plasma membranes where they form a complex with cytochrome b(558) and express enzyme activity. A 29-kDa neutrophil protein (p29) was identified by co-immunoprecipitation with p67(phox). N-terminal sequence analysis of p29 revealed homology to an open reading frame gene described in a myeloid leukemia cell line. A cDNA for p29 identical to the open reading frame protein was amplified from RNA of neutrophils. Significant interaction between p29 and p67(phox) was demonstrated using a yeast two-hybrid system. A recombinant (rh) p29 was expressed in Sf9 cells resulting in a protein with an apparent molecular weight of 34,000. The rh-p29 showed immunoreactivity with the original rabbit antiserum that detected p47(phox) and p67(phox). In addition, rh-p29 exhibited PLA(2) activity, which was Ca(2+) independent, optimal at low pH, and preferential for phosphatidylcholine substrates. The recombinant protein protected glutathione synthetase and directly inactivated H(2)O(2). By activity and sequence homology, rh-p29 can be classified as a peroxiredoxin. Finally, O(2)() production by plasma membrane and recombinant cytosolic oxidase components in the SDS-activated, cell-free NADPH oxidase system were enhanced by rh-p29. This effect was not inhibited by PLA(2) inhibitors. Thus, p29 is a novel protein that associates with p67 and has peroxiredoxin activity. This protein has a potential role in protecting the NADPH oxidase by inactivating H(2)O(2) or altering signaling pathways affected by H(2)O(2).
毒性氧代谢产物的产生为中性粒细胞的杀菌活性提供了一种机制。NADPH氧化酶系统通过将氧还原形成超氧阴离子（O₂⁻）来启动氧代谢产物的产生。随着呼吸爆发的刺激，胞质氧化酶成分p47⁽ᵖʰᵒˣ⁾、p67⁽ᵖʰᵒˣ⁾和Rac易位至吞噬溶酶体膜和质膜，在那里它们与细胞色素b₅₅₈形成复合物并表达酶活性。通过与p67⁽ᵖʰᵒˣ⁾的共免疫沉淀鉴定出一种29 kDa的中性粒细胞蛋白（p29）。p29的N端序列分析显示与髓系白血病细胞系中描述的一个开放阅读框基因具有同源性。从嗜中性粒细胞的RNA中扩增出与开放阅读框蛋白相同的p29的cDNA。使用酵母双杂交系统证明了p29与p67⁽ᵖʰᵒˣ⁾之间存在显著相互作用。重组（rh）p29在Sf9细胞中表达产生一种表观分子量为34000的蛋白质。rh - p29与检测p47⁽ᵖʰᵒˣ⁾和p67⁽ᵖʰᵒˣ⁾的原始兔抗血清表现出免疫反应性。此外，rh - p29表现出磷脂酶A₂（PLA₂）活性，该活性不依赖Ca²⁺，在低pH下最佳，且对磷脂酰胆碱底物具有偏好性。重组蛋白保护谷胱甘肽合成酶并直接使H₂O₂失活。通过活性和序列同源性，rh - p29可归类为过氧化物还原酶。最后，在SDS激活的无细胞NADPH氧化酶系统中，rh - p29增强了质膜和重组胞质氧化酶成分产生的O₂⁻。这种作用不受PLA₂抑制剂的抑制。因此，p29是一种与p67相关的新型蛋白，具有过氧化物还原酶活性。该蛋白在通过使H₂O₂失活或改变受H₂O₂影响的信号通路来保护NADPH氧化酶方面具有潜在作用。
