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

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 Corthé sy, B. (1999) All three loops, referred to as B-C, D-E, and F-G in domain I are particularly implicated in binding to IgA (4, 5); in addition, some residues of loop E-F in the opposite face appear to be important for IgA binding. In contrast, deletion of domains II and III or individual deletion of domain IV and V of rabbit pIgR did not prevent binding to IgA in a qualitative cell-ligand binding assay (5). A natural variant of rabbit SC lacking domains II and III can bind to IgA, although only in a noncovalent fashion (6, 7). In human sIgA, a disulfide bridge within domain V is also involved in covalent binding to the C␣2 domain of IgA through a disulfide exchange mechanism (8 -10). Species variations in the level of covalency between pIgR and IgA have been reported (11,12). Thus, it is tempting to speculate that although not critical for the initial recognition of IgA, domains II to IV appear to position domain V such that disulfide exchange with IgA can take place.The mechanism for the selective recognition of IgA d over IgA m remains an additional open question. We have generated murine SC mutants with predicted exposed loops of domains I, II, and III replaced with the FLAG epitope (13) to evaluate their IgA binding properties. We show that although the affinity of these mutants for IgA and the selectivity for IgA d over IgA m are both preserved, covalent binding is lost in all mutants but one. Given the pinpointing strategy designed here, this suggests that domains II and III are essential to the proper positioning of domain V. However, because binding to the anti-FLAG mAb is preserved in both free and IgA d -bound reactive SC mutants, we postulate that minor structural changes occur upon IgA d -mSC interactions. Dimeric SC-FLAG mutants were shown to be as good IgA d binders as their monomeric counterparts. In the same assay, we found by direct comparison of mSC and pIgR mutants that mSC binds to IgA d with much reduced stringency. We conclude that mSC-FLAG mutants are useful 1) to study the topology of SC⅐IgA d complexes, 2) as short epitope carrier once combined with IgA d , 3) to tackle the binding specificity and stoichiometry of SC/pIgR IgA d complexes.

The Nonplanar Secretory IgA2 and Near Planar Secretory IgA1 Solution Structures Rationalize Their Different Mucosal Immune Responses

“…8C). Thus the SIgA2 structure is consistent with the proposal for SIgA1 in which a "zipper effect" association of the unbound J-shaped SC domain structure occurs along the edge of the Fc regions (20,46). The lack of the extended hinge in SIgA2 does, of course, mean that SIgA2 cannot perform the functions mediated by the O-linked sugars of the IgA1 hinge that serve as ligands for cellular receptors and bind pathogens such as S-fimbriated Escherichia coli (47,48).…”

Glycans on Secretory Component Participate in Innate Protection against Mucosal Pathogens

“…Because EPEC adhesion molecule intimin possesses Ig-like domains and a terminal C-type lectin-like domain (29, 30), we postulated that (i) such domains might associate with similar domains (31)(32)(33) and/or glycans displayed by hSC and (ii) such an interaction would alter the intimin-dependent EPEC adhesion as reflected by the formation of A/E lesions (34). In preliminary in vitro binding experiments, we observed that in the presence of hSCrec, the adhesion of wild type EPEC to target HEp-2 cells was reduced to levels comparable with those found with the int Ϫ EPEC mutant (Fig.…”

Activity of Human IgG and IgA Subclasses in Immune Defense Against Neisseria meningitidis Serogroup B

“…The "docking site" on IgA for Fc␣RI has been mapped to the boundary of CH2 and CH3 (38,39), a location occupied by SC (40). To evaluate the effect of SC on IgA-Fc␣RI binding, we fractionated IgA1 isolated from supernatants of IgA1-J-SC and that of IgA2-J-transfected BHK cells.…”