Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome.
OBJECTIVE
Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity-determining regions (CDRs) of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in V(H) CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 V(H).


METHODS
R53 was altered, by site-directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of V(H). In addition, the germline sequence of the V(H)3-23 gene (from which B3 V(H) is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild-type B3 V(H), were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, alpha-actinin, cardiolipin, ovalbumin, beta(2)-glycoprotein I (beta(2)GPI), and the N-terminal domain of beta(2)GPI (domain I), using direct binding assays.


RESULTS
The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to alpha-actinin, ovalbumin, beta(2)GPI, and domain I of beta(2)GPI. The combination B3 (R53S) V(H)/B3 V(L) bound human, but not bovine, beta(2)GPI.


CONCLUSIONS
The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3.
Lambrianides
Anastasia
A
University College London, London, UK.
Giles
Ian
I
Ioannou
Yiannis
Y
Mason
Lesley
L
Latchman
David S
DS
Manson
Jessica J
JJ
Isenberg
David A
DA
Rahman
Anisur
A
D000595:Amino Acid Sequence; D016736:Antiphospholipid Syndrome / Q000235:genetics* / Q000276:immunology; D001120:Arginine; D001665:Binding Sites; D017931:DNA Primers; D018095:Germ-Line Mutation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008180:Lupus Erythematosus, Systemic / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010047:Ovalbumin / Q000378:metabolism; D015203:Reproducibility of Results
D017931:DNA Primers; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010047:Ovalbumin; D001120:Arginine