Relative importance of different human aPL derived heavy and light chains in the binding of aPL to cardiolipin.
BACKGROUND
Previous studies have suggested the importance of somatic mutations and arginine, asparagine and lysine residues in the complementarity determining regions (CDRs) of antiphospholipid antibodies (aPL) implicated in the pathogenesis of the antiphospholipid antibody syndrome. The relative contributions of the heavy and light chains of aPL in binding to cardiolipin (CL) were assessed by pairing the heavy and light chains of two IgG, beta(2)GPI dependent aPL (IS4 and CL24) with different partner chains from other IgG, beta(2)GPI independent aPL (UK4) and anti-DNA antibodies (B3 and 33H11).


METHODS
Four heavy (V(H)) and five light (V(L)) chain variable sequences from three aPL and two anti-DNA antibodies were cloned into expression vectors containing appropriate gamma(1), lambda or kappa constant region cDNA. Paired combinations of heavy and light chain expression plasmids were transfected into COS-7 cells allowing transient expression of whole IgG molecules, which were harvested and tested for the ability to bind CL and DNA by enzyme-linked immunosorbant assay (ELISA).


RESULTS
Whole IgG was produced from 19 heavy/light chain combinations. IS4V(H) was dominant in conferring the ability to bind CL with four of the five V(L) tested. The identity of the V(L) region paired with IS4V(H) was important in determining the strength of binding to CL. IS4V(H) contains multiple arginine residues in CDR3, which may have accumulated due to antigen driven selection. It is likely that these arginine residues may interact with CL. The combination B3V(H)/B3V(L) also bound CL, but none of the other 14 combinations showed any binding in this assay.


CONCLUSIONS
Whole IgG molecules capable of binding CL were produced by in vitro expression in COS-7 cells. Arginine residues play important roles in binding to CL and double-stranded DNA. However, different patterns of mutation to arginine are associated with binding to each of these antigens.
Giles
Ian P
IP
Department of Medicine, Centre for Rheumatology, University College London, Arthur Stanley House, 40-50 Tottenham Street, London W1T 4NJ, UK. i.giles@ich.ucl.ac.uk
Haley
Joanna
J
Nagl
Sylvia
S
Latchman
David S
DS
Chen
Pojen P
PP
Chukwuocha
Reginald U
RU
Isenberg
David A
DA
Rahman
Anisur
A
D000595:Amino Acid Sequence; D000818:Animals; D017152:Antibodies, Antiphospholipid / Q000737:chemistry* / Q000378:metabolism; D019556:COS Cells; D002308:Cardiolipins / Q000378:metabolism*; D022801:Complementarity Determining Regions; D004247:DNA / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D053482:beta 2-Glycoprotein I
D017152:Antibodies, Antiphospholipid; D002308:Cardiolipins; D022801:Complementarity Determining Regions; D006023:Glycoproteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D053482:beta 2-Glycoprotein I; D004247:DNA