Abstract
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.
Highlights
The first line of defense against pathogens consists of mucosal secretions, with secretory IgA being one of the main effectors
1 The abbreviations used are: pIgR, polymeric immunoglobulin receptor; mpIgR, murine pIgR; IgAd, dimeric IgA; IgAm, monomeric IgA; IgAp, polymeric IgA; sIgA, secretory IgA; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; SC, secretory component; hSC, human SC; mSC, murine SC; IC50, 50% inhibitory concentration; mAb, monoclonal antibody; PBS, phosphate-buffered saline; pIgR is expressed by epithelial cells in a variety of mucosal tissues and in rodents is further implicated in transport into bile through the liver [1]
This is the first report studying the interaction between murine IgA and murine SC
Summary
The structure and production of the various recombinant proteins and most analytical procedures as well as the source of most antibodies and reagents are described in the accompanying article [13]. 50 l of biotinylated IgG was applied to native mSC (40 g/ml, protein A, purified) for 1 h at 4 °C and detected with HRP-coupled ExtrAvidin at 1:1,000 dilution. Murine SC1⁄7IgA Co-immunoprecipitation—Identical amounts (corresponding to 200 ng of protein) of mSC mutants purified by concanavalin A-agarose chromatography were incubated with 1 g of biotinylated IgAd in 50 l of PBS for 1 h at room temperature. Such conditions have been shown to permit full covalent association of SC1⁄7IgAd in vitro [16].
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