Bergt C, Oettl K, Keller W, Andreae F, Leis H J, Malle E, Sattler W
Institute of Medical Biochemistry, University Graz, Austria.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):345-54.
We have previously shown that the modification of high-density lipoprotein subclass 3 (HDL(3)) by HOCl transformed an anti-atherogenic lipoprotein into a high-uptake form for macrophages and caused a significant impairment of cholesterol efflux capacity [Panzenboeck, Raitmayer, Reicher, Lindner, Glatter, Malle and Sattler (1997) J. Biol. Chem. 272, 29711-29720]. To elucidate the consequences of treatment with OCl(-) on distinct regions in apolipoprotein A-I (apo A-I), lipid-free and lipid-associated apo A-I were modified with increasing molar ratios of NaOCl or HOCl generated by the myeloperoxidase/H(2)O(2)/Cl(-) system. CD analysis revealed a pronounced decrease in alpha-helicity for lipid-free apo A-I modified by NaOCl, whereas lipid-associated apo A-I was less affected. The modification of apo A-I by NaOCl (molar oxidant-to-lipoprotein ratio 6:1) resulted in the formation of two distinct oxidized forms of apo A-I with molecular masses 32 or 48 atomic mass units (a.m.u.) higher than that of native apo A-I, indicating the addition of two or three oxygen atoms to the native protein. HPLC analysis of tryptic digests obtained from lipid-free and lipid-associated apo A-I modified with increasing oxidant-to-apolipoprotein molar ratios revealed a concentration-dependent modification of apo A-I: at a low molar oxidant-to-lipoprotein ratio (5:1) the peaks corresponding to the methionine-containing tryptic peptides T11 (residues 84-88), T16 (residues 108-116) and T22 (residues 141-149), located in the central region of apo A-I, disappeared. Their loss was accompanied by the formation of three oxidation products with a molecular mass 16 a.m.u. higher than that of the native peptides. This indicates the addition of oxygen, most probably caused by the oxidation of Met(86), Met(112) and Met(148) to the corresponding methionine sulphoxides. At a molar NaOCl-to-apo A-I ratio of 10:1 the disappearance of peptides T1 (residues 1-10), T7 (residues 46-59) and T9 (residues 62-77) was accompanied by the occurrence of new peaks 33.5 and 33.1 a.m.u. higher than those of the native peptides. Amino acid analyses of peptides T7 and T9 after modification with NaOCl confirmed that Phe(57) and Phe(71) were primary targets for oxidation by HOCl. GLC-MS analysis of hydrolysates obtained from OCl(-)-modified T7, T9, apo A-I and HDL(3) confirmed that Phe residues are an early target for OCl(-) modification. At molar NaOCl-to-apo A-I ratios of 25:1, the peak areas of peptides T31 (residues 189-195) and T32 (residues 196-206) decreased markedly. Most importantly, incubation of apo A-I with the myeloperoxidase/H(2)O(2)/Cl(-) system (the source of HOCl in vivo) resulted in almost identical modification patterns to those observed with reagent NaOCl.
我们之前已经表明，次氯酸（HOCl）对高密度脂蛋白亚类3（HDL(3)）的修饰将一种抗动脉粥样硬化脂蛋白转变为巨噬细胞的高摄取形式，并导致胆固醇流出能力显著受损[Panzenboeck、Raitmayer、Reicher、Lindner、Glatter、Malle和Sattler（1997年）《生物化学杂志》272，29711 - 29720]。为了阐明用次氯酸根离子（OCl(-)）处理对载脂蛋白A - I（apo A - I）不同区域的影响，将无脂和与脂质结合的apo A - I用髓过氧化物酶/H(2)O(2)/Cl(-)系统产生的NaOCl或HOCl的摩尔比增加进行修饰。圆二色性（CD）分析显示，NaOCl修饰的无脂apo A - I的α螺旋度显著降低，而与脂质结合的apo A - I受影响较小。NaOCl（氧化剂与脂蛋白的摩尔比为6:1）对apo A - I的修饰导致形成两种不同的氧化形式的apo A - I，其分子量比天然apo A - I高32或48原子质量单位（a.m.u.），表明在天然蛋白质上添加了两个或三个氧原子。对用增加的氧化剂与载脂蛋白摩尔比修饰的无脂和与脂质结合的apo A - I的胰蛋白酶消化物进行高效液相色谱（HPLC）分析，显示apo A - I存在浓度依赖性修饰：在低氧化剂与脂蛋白摩尔比（5:1）时，对应于位于apo A - I中心区域的含甲硫氨酸的胰蛋白酶肽T11（残基84 - 88）、T16（残基108 - 116）和T22（残基141 - 149）的峰消失。它们的消失伴随着形成三种分子量比天然肽高16 a.m.u.的氧化产物。这表明添加了氧，很可能是由于Met(86)、Met(112)和Met(148)氧化为相应的甲硫氨酸亚砜所致。在NaOCl与apo A - I的摩尔比为10:1时，肽T1（残基1 - 10）、T7（残基46 - 59）和T9（残基62 - 77）的消失伴随着出现比天然肽高33.5和33.1 a.m.u.的新峰。用NaOCl修饰后对肽T7和T9进行氨基酸分析证实，Phe(57)和Phe(71)是HOCl氧化的主要靶点。对从OCl(-)修饰的T7、T9、apo A - I和HDL(3)获得的水解产物进行气相色谱 - 质谱（GLC - MS）分析证实，苯丙氨酸残基是OCl(-)修饰的早期靶点。在NaOCl与apo A - I的摩尔比为25:1时，肽T31（残基189 - 195）和T32（残基196 - 206）的峰面积显著降低。最重要的是，将apo A - I与髓过氧化物酶/H(2)O(2)/Cl(-)系统（体内HOCl的来源）孵育导致的修饰模式与用试剂NaOCl观察到的几乎相同。
