Mapping the interaction between murine IgA and murine secretory component carrying epitope substitutions reveals a role of domains II and III in covalent binding to IgA.
We have identified sites for epitope insertion in the murine secretory component (SC) by replacing individual surface-exposed loops in domains I, II, and III with the FLAG sequence (Crottet, P., Peitsch, M. C., Servis, C., and Corthésy, B. (1999) J. Biol. Chem. 274, 31445-31455). We had previously shown that epitope-carrying SC reassociated with dimeric IgA (IgA(d)) can serve as a mucosal delivery vehicle. When analyzing the capacity of SC mutants to associate with IgA(d), we found that all domain II and III mutants bound specifically with immobilized IgA(d), and their affinity for IgA(d) was comparable to that of the wild type protein (IC(50) approximately 1 nM). We conclude that domains II and III in SC are permissive to local mutation and represent convenient sites to antigenize the SC molecule. No mutant bound to monomeric IgA. SC mutants exposing the FLAG at their surface maintained this property once bound to IgA(d), thereby defining regions not required for high affinity binding to IgA(d). Association of IgA(d) with SC mutants carrying a buried FLAG did not expose de novo the epitope, consistent with limited, local changes in the SC structure upon binding. Only wild type and two mutant SCs bound covalently to IgA(d), thus implicating domains II and III in the correct positioning of the reactive cysteine in SC. This establishes that the integrity of murine SC domains II and III is not essential to preserve specific IgA(d) binding but is necessary for covalency to take place. Finally, SC mutants existing in the monomeric and dimeric forms exhibited the same IgA(d) binding capacity as monomeric wild type SC known to bind with a 1:1 stoichiometry.
Crottet
P
P
Institut Suisse de Recherches Expérimentales sur le Cancer, CH-1066 Epalinges, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland.
Corthésy
B
B
D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D000939:Epitopes; D007070:Immunoglobulin A / Q000378:metabolism; D007071:Immunoglobulin A, Secretory / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides; D010455:Peptides; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019056:Receptors, Polymeric Immunoglobulin / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D007070:Immunoglobulin A; D007071:Immunoglobulin A, Secretory; D009842:Oligopeptides; D010455:Peptides; D019056:Receptors, Polymeric Immunoglobulin; C034639:polymeric IgA; C085706:FLAG peptide