Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a).
Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.
Edelstein
Celina
C
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Pfaffinger
Ditta
D
Hinman
Janet
J
Miller
Elizabeth
E
Lipkind
Gregory
G
Tsimikas
Sotirios
S
Bergmark
Claes
C
Getz
Godfrey S
GS
Witztum
Joseph L
JL
Scanu
Angelo M
AM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001054:Apolipoproteins A / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008163:Luminescent Measurements; D008239:Lysine / Q000737:chemistry*; D008253:Macaca mulatta; D008264:Macrophages / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010758:Phosphorus / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D001054:Apolipoproteins A; D017077:Culture Media, Conditioned; D016209:Interleukin-8; D008055:Lipids; D010713:Phosphatidylcholines; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010758:Phosphorus; D008239:Lysine; D010100:Oxygen