Covalent homodimers of murine secretory component induced by epitope substitution unravel the capacity of the polymeric Ig receptor to dimerize noncovalently in the absence of IgA ligand.
Recombinant secretory immunoglobulin A containing a bacterial epitope in domain I of the secretory component (SC) moiety can serve as a mucosal delivery vehicle triggering both mucosal and systemic responses (Corthésy, B., Kaufmann, M., Phalipon, A., Peitsch, M., Neutra, M. R., and Kraehenbuhl, J.-P. (1996) J. Biol. Chem. 271, 33670-33677). To load recombinant secretory IgA with multiple B and T epitopes and extend its biological functions, we selected, based on molecular modeling, five surface-exposed sites in domains II and III of murine SC. Loops predicted to be exposed at the surface of SC domains were replaced with the DYKDDDDK octapeptide (FLAG). Another two mutants were obtained with the FLAG inserted in between domains II and III or at the carboxyl terminus of SC. As shown by mass spectrometry, internal substitution of the FLAG into four of the mutants induced the formation of disulfide-linked homodimers. Three of the dimers and two of the monomers from SC mutants could be affinity-purified using an antibody to the FLAG, mapping them as candidates for insertion. FLAG-induced dimerization also occurred with the polymeric immunoglobulin receptor (pIgR) and might reflect the so-far nondemonstrated capacity of the receptor to oligomerize. By co-expressing in COS-7 cells and epithelial Caco-2 cells two pIgR constructs tagged at the carboxyl terminus with hexahistidine or FLAG, we provide the strongest evidence reported to date that the pIgR dimerizes noncovalently in the plasma membrane in the absence of polymeric IgA ligand. The implication of this finding is discussed in terms of IgA transport and specific antibody response at mucosal surfaces.
Crottet
P
P
Institut Suisse de Recherches, Expérimentales sur le Cancer, CH-1066 Epalinges, Switzerland.
Peitsch
M C
MC
Servis
C
C
Corthésy
B
B
D000818:Animals; D019556:COS Cells; D018938:Caco-2 Cells; D002462:Cell Membrane; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000378:metabolism*; D007071:Immunoglobulin A, Secretory / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D009842:Oligopeptides; D010455:Peptides; D011233:Precipitin Tests; D015202:Protein Engineering; D019056:Receptors, Polymeric Immunoglobulin / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000939:Epitopes; D018796:Immunoconjugates; D007071:Immunoglobulin A, Secretory; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D019056:Receptors, Polymeric Immunoglobulin; C085706:FLAG peptide