Reagent or myeloperoxidase-generated hypochlorite affects discrete regions in lipid-free and lipid-associated human apolipoprotein A-I.
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.
Bergt
C
C
Institute of Medical Biochemistry, University Graz, Austria.
Oettl
K
K
Keller
W
W
Andreae
F
F
Leis
H J
HJ
Malle
E
E
Sattler
W
W
D016632:Apolipoprotein A-I / Q000737:chemistry*; D002712:Chlorides / Q000737:chemistry; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D009195:Peroxidase / Q000737:chemistry*; D017510:Protein Folding
D016632:Apolipoprotein A-I; D002712:Chlorides; D008055:Lipids; D009195:Peroxidase; C001599:chlorite