The Carboxyl-terminal Domains of IgA and IgM Direct Isotype-specific Polymerization and Interaction with the Polymeric Immunoglobulin Receptor
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m205502200
ISSN1083-351X
AutoresRanveig Braathen, Vigdis Sörensen, Per Brandtzæg, Inger Sandlie, Finn-Eirik Johansen,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoMucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Cα3 and Cμ4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Cα3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component. Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Cα3 and Cμ4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Cα3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component. polymeric IgA polymeric Ig receptor secretory IgA joining chain secretory component 5-iodo-4-hydroxy-3-nitrophenylacetyl enzyme-linked immunosorbent assay phosphate-buffered saline Madin-Darby canine kidney monoclonal antibody bovine serum albumin At least 80% of all antibody-secreting plasma cells of the body are located in the gastrointestinal and respiratory mucosae, and most of them are committed to immunoglobulin A (IgA) production (1Brandtzaeg P. Farstad I.N. Johansen F.E. Morton H.C. Norderhaug I.N. Yamanaka T. Immunol. Rev. 1999; 171: 45-87Crossref PubMed Scopus (236) Google Scholar, 2Norderhaug I.N. Johansen F.E. Schjerven H. Brandtzaeg P. Crit. Rev. Immunol. 1999; 19: 481-508PubMed Google Scholar). All immunoglobulin isotypes consists of two heavy (H) and two light (L) chains, but for IgA, this H2L2 monomeric unit can polymerize further. Mucosally produced IgA consists predominantly of dimers and some larger polymers, collectively called polymeric IgA (pIgA).1 IgA polymerization is regulated by the incorporation of the joining chain (J chain) in that its presence greatly stimulates polymerization (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar, 4Vaerman J.P. Langendries A. Vander Maelen C. Immunol. Invest. 1995; 24: 631-641Crossref PubMed Scopus (35) Google Scholar, 5Johansen F.E. Braathen R. Brandtzaeg P. J. Immunol. 2001; 167: 5185-5192Crossref PubMed Scopus (166) Google Scholar, 6Johansen F.E. Natvig Norderhaug I. Røe M. Sandlie I. Brandtzaeg P. Eur. J. Immunol. 1999; 29: 1701-1708Crossref PubMed Scopus (42) Google Scholar, 7Ma J.K. Hiatt A. Hein M. Vine N.D. Wang F. Stabila P. van Dolleweerd C. Mostov K. Lehner T. Science. 1995; 268: 716-719Crossref PubMed Scopus (473) Google Scholar, 8Carayannopoulos L. Max E.E. Capra J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8348-8352Crossref PubMed Scopus (77) Google Scholar, 9Krugmann S. Pleass R.J. Atkin J.D. Woof J.M. J. Immunol. 1997; 159: 244-249PubMed Google Scholar). J chain is also essential in forming a docking site on pIgA for the polymeric immunoglobulin receptor (pIgR) (5Johansen F.E. Braathen R. Brandtzaeg P. J. Immunol. 2001; 167: 5185-5192Crossref PubMed Scopus (166) Google Scholar, 10Brandtzaeg P. Scand. J. Immunol. 1975; 4: 837-842Crossref PubMed Google Scholar, 11Brandtzaeg P. Prydz H. Nature. 1984; 311: 71-73Crossref PubMed Scopus (327) Google Scholar, 12Johansen F.E. Braathen R. Brandtzaeg P. Scand. J. Immunol. 2000; 52: 240-248Crossref PubMed Scopus (214) Google Scholar, 13Vaerman J.P. Langendries A. Giffroy D. Brandtzaeg P. Kobayashi K. Immunology. 1998; 95: 90-96Crossref PubMed Scopus (43) Google Scholar, 14Vaerman J.P. Langendries A.E. Giffroy D.A. Kaetzel C.S. Fiani C.M. Moro I. Brandtzaeg P. Kobayashi K. Eur. J. Immunol. 1998; 28: 171-182Crossref PubMed Scopus (39) Google Scholar). This 110-kDa glycoprotein binds pIgA at the basolateral epithelial cell surface. Receptor-IgA complexes are internalized and then transcytosed to the apical surface, where secretory IgA (S-IgA) is released into the lumen by proteolytic cleavage of the receptor ectodomain (2Norderhaug I.N. Johansen F.E. Schjerven H. Brandtzaeg P. Crit. Rev. Immunol. 1999; 19: 481-508PubMed Google Scholar, 15Mostov K. Kaetzel C.S. Ogra P.L. Mestecky J. Lamm M.E. Strober W. Bienenstock J. McGhee J.R. Mucosal Immunology. Academic Press, Inc., San Diego1999: 181-211Google Scholar). Cleavage of unoccupied receptor releases the five extracellular immunoglobulin-like domains (D1–D5), known as free secretory component (SC).IgM also has the ability to polymerize, mainly to pentamers with incorporated J chain and to hexamers without (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar, 16Wiersma E.J. Collins C. Fazel S. Shulman M.J. J. Immunol. 1998; 160: 5979-5989PubMed Google Scholar, 17Brewer J.W. Corley R.B. Mol. Immunol. 1997; 34: 323-331Crossref PubMed Scopus (34) Google Scholar). As for IgA, only J chain-containing IgM polymers bind the pIgR (11Brandtzaeg P. Prydz H. Nature. 1984; 311: 71-73Crossref PubMed Scopus (327) Google Scholar, 18Eskeland T. Brandtzaeg P. Immunochemistry. 1974; 11: 161-163Crossref PubMed Scopus (77) Google Scholar, 19Mach J.P. Nature. 1970; 228: 1278-1282Crossref PubMed Scopus (95) Google Scholar, 20Brandtzaeg P. Scand. J. Immunol. 1976; 5: 411-419Crossref PubMed Scopus (60) Google Scholar). IgM is believed to be ancestral to all immunoglobulin classes (21Warr G.W. Magor K.E. Higgins D.A. Immunol. Today. 1995; 16: 392-398Abstract Full Text PDF PubMed Scopus (379) Google Scholar). During evolution of IgG, the ability to form polymers, incorporate the J chain, and bind to the pIgR was lost. Thus, the IgM and IgA heavy chains (μ- and α-chain, respectively) have a number of features that are absent in IgG. Both IgM and IgA have a COOH-terminal extension of 18 amino acids called the secretory tailpiece, which includes a cysteine required for polymerization in the penultimate position (Cys-575 in μ tailpiece; Cys-495 in α tailpiece). Besides the secretory tailpiece cysteines, Cys-337 and Cys-414 are available for disulfide bonding in IgM. Whereas Cys-337 most likely forms intramonomeric bonds between two Cμ2 domains, Cys-414 (located in Cμ3) forms intermonomeric bonds (22Beale D. Feinstein A. Biochem. J. 1969; 112: 187-194Crossref PubMed Scopus (47) Google Scholar, 23Davis A.C. Roux K.H. Pursey J. Shulman M.J. EMBO J. 1989; 8: 2519-2526Crossref PubMed Scopus (86) Google Scholar, 24Sitia R. Neuberger M. Alberini C. Bet P. Fra A. Valetti C. Williams G. Milstein C. Cell. 1990; 60: 781-790Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 25Wiersma E.J. Shulman M.J. J. Immunol. 1995; 154: 5265-5272PubMed Google Scholar, 26Milstein C.P. Richardson N.E. Dieverson E.V. Feinstein A. Biochem. J. 1975; 151: 615-624Crossref PubMed Scopus (37) Google Scholar). Conversely, Cys-309 in IgA (equivalent to Cys-414 in IgM) is used for covalent bonding to pIgR during transcytosis (27Fallgreen-Gebauer E. Gebauer W. Bastian A. Kratzin H.D. Eiffert H. Zimmermann B. Karas M. Hilschmann N. Biol. Chem. Hoppe. Seyler. 1993; 374: 1023-1028Crossref PubMed Scopus (57) Google Scholar). Moreover, during evolution of IgA the polymerization process has altered to favor dimerization rather than formation of larger polymers. Monomers are also secreted from IgA-producing plasma cells, whereas they are mainly retained and degraded in IgM-producing plasma cells (24Sitia R. Neuberger M. Alberini C. Bet P. Fra A. Valetti C. Williams G. Milstein C. Cell. 1990; 60: 781-790Abstract Full Text PDF PubMed Scopus (194) Google Scholar).J chain incorporation is an early event in IgA polymerization, and this peptide is found in all polymeric forms of this isotype (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar,4Vaerman J.P. Langendries A. Vander Maelen C. Immunol. Invest. 1995; 24: 631-641Crossref PubMed Scopus (35) Google Scholar). For IgM, however, the J chain is incorporated late in the polymerization process and is found only in pentamers (17Brewer J.W. Corley R.B. Mol. Immunol. 1997; 34: 323-331Crossref PubMed Scopus (34) Google Scholar). Furthermore, the mode of pIgR binding differs significantly between the two isotypes, and in some species (most notably the rabbit) the ability of the pIgR to bind pentameric IgM has been selectively lost (28Røe M. Norderhaug I.N. Brandtzaeg P. Johansen F.E. J. Immunol. 1999; 162: 6046-6052PubMed Google Scholar, 29Weicker J. Underdown B.J. J. Immunol. 1975; 114: 1337-1344PubMed Google Scholar). To address the structural basis for the disparate polymerization properties of IgA and IgM and their differential mode of interaction with pIgR, we made a series of domain swap mutants between human IgA1 and human IgM. Our results demonstrate that the COOH-terminal domains of IgA and IgM contain the most important structural elements involved in isotype-specific polymerization. We also found that swapping Cμ4 with Cα3 made IgM able to bind to the rabbit pIgR, indicating that the COOH-terminal domain is also most important for the differential receptor binding.DISCUSSIONIgA and IgM have unique motifs, not found in IgG, which allow them to form polymers and incorporate J chain. Such J chain-containing polymers are selectively transported by pIgR into exocrine fluids to form secretory antibodies (S-IgA and S-IgM). The extensive antigenic exposure of the mucosae underscores the need for a specific first-line defense. However, differences between IgA and IgM exist, both in the size of polymers formed and in their J chain incorporation and mode of interaction with the pIgR. To localize structural motifs that determine these differences, we made domain swap mutants between IgA and IgM heavy chains. The results clearly showed that the COOH-terminal domains (Cα3 and Cμ4) primarily direct the degree of polymer formation. Furthermore, we found that J chain incorporation was restricted to immunoglobulin pentamers containing Cμ4 but occurred in all polymeric forms of immunoglobulins containing Cα3. SC binding and pIgR-mediated transcytosis also demonstrated that the COOH-terminal domains contained the structural elements that determine differential interaction of J chain-containing pIgA and pentameric IgM with rabbit and human pIgR/free SC. However, the COOH-terminal domain was not the only determinant contributing to the functional properties of the chimeric immunoglobulins, because the pentameric AAM chimera showed increased binding to human pIgR/free SC compared with both IgM and IgA and a similar degree of covalent SC complexing as IgA.Structural Requirements for Immunoglobulin PolymerizationAlthough the structures of the α- and μ-chains clearly differ, the exact motifs directing the differential polymerization patterns observed for IgA and IgM remain elusive. We (30Sørensen V. Rasmussen I.B. Norderhaug L. Natvig I. Michaelsen T.E. Sandlie I. J. Immunol. 1996; 156: 2858-2865PubMed Google Scholar, 37Sørensen V. Sundvold V. Michaelsen T.E. Sandlie I. J. Immunol. 1999; 162: 3448-3455PubMed Google Scholar) and others (35Smith R.I. Coloma M.J. Morrison S.L. J. Immunol. 1995; 154: 2226-2236PubMed Google Scholar, 36Smith R.I. Morrison S.L. Bio/Technology. 1994; 12: 683-688Crossref PubMed Scopus (39) Google Scholar, 38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) have previously investigated the secretory tailpiece sequences unique to polymeric immunoglobulins. Recombinant human IgM engineered to contain an α tailpiece (IgMαtp) was found to polymerize like IgM, although with increased hexamer formation (30Sørensen V. Rasmussen I.B. Norderhaug L. Natvig I. Michaelsen T.E. Sandlie I. J. Immunol. 1996; 156: 2858-2865PubMed Google Scholar), whereas the reciprocal mutation with μ tailpiece introduced into IgA led to the formation of some polymers larger than wild-type pIgA (37Sørensen V. Sundvold V. Michaelsen T.E. Sandlie I. J. Immunol. 1999; 162: 3448-3455PubMed Google Scholar). Both μ-tailpiece and α-tailpiece sequences induced formation of polymers including pentamers and hexamers when added onto IgG, and such polymerization was most efficient when the secretory tailpiece was introduced in conjunction with a Cys-414/Cys-309 homologue in Cγ2 (30Sørensen V. Rasmussen I.B. Norderhaug L. Natvig I. Michaelsen T.E. Sandlie I. J. Immunol. 1996; 156: 2858-2865PubMed Google Scholar, 35Smith R.I. Coloma M.J. Morrison S.L. J. Immunol. 1995; 154: 2226-2236PubMed Google Scholar, 36Smith R.I. Morrison S.L. Bio/Technology. 1994; 12: 683-688Crossref PubMed Scopus (39) Google Scholar, 38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These results suggested that although the secretory tailpiece is sufficient to drive the polymerization process, it does not by itself direct the number of monomers incorporated into the polymers. The analyses of the domain swap mutants described here were carried out to identify the constant region domain(s) that harbors the elements required for isotype-specific polymerization and pIgR-binding properties of polymeric immunoglobulins.Together with the light chain and the heavy chain variable domain, the first constant domain forms the so-called Fab portion of the immunoglobulin. The remainder of the heavy chain forms the so-called Fc portion believed to be responsible for most isotype-specific effector functions. To test whether the first constant domain could affect the polymerization pattern of the chimeric immunoglobulins, we exchanged the Cμ1 domain in IgM with the Cα1 domain, producing the AMMM chimera. We also made the same substitution in the MMMA chimera producing the AMMA variant. In both cases, the polymerization pattern of the resulting chimera was nearly identical to that of the parental immunoglobulin. Thus, our findings indicated that the first heavy chain domain does not influence the number of monomers linked during polymerization. This conclusion is supported by the observations that immunoglobulin light chains, which are normally bound to the Cμ1 domain, are not required for efficient polymerization of IgM (45Bornemann K.D. Brewer J.W. Beck-Engeser G.B. Corley R.B. Haas I.G. Jack H.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4912-4916Crossref PubMed Scopus (22) Google Scholar) and that IgM lacking the Cμ1 domain is also secreted as polymers (46Kohler G. Potash M.J. Lehrach H. Shulman M.J. EMBO J. 1982; 1: 555-563Crossref PubMed Scopus (71) Google Scholar).Other studies have suggested that the Cμ2 region is not essential for IgM pentamer assembly, but the Cμ3 domain may play an important role in the polymerization (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar, 47Poon P.H. Morrison S.L. Schumaker V.N. J. Biol. Chem. 1995; 270: 8571-8577Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). We have previously reported that mutation of five amino acids flanking Cys-309 in IgA into the corresponding amino acids present in IgM, forming the so-called IgAalm mutant, resulted in only a small increase of trimers and tetramers (37Sørensen V. Sundvold V. Michaelsen T.E. Sandlie I. J. Immunol. 1999; 162: 3448-3455PubMed Google Scholar). The AMMA variant, which contained Cμ2 and Cμ3, formed some larger polymers as compared with IgA and the IgAalm mutant. Thus, other structural motifs in Cμ3 may be more important in pentamer formation than the Cys-309 region.We identified the Cα3 and Cμ4 domains as most important for isotype-specific polymerization. Whereas an IgA-like polymerization pattern was observed for the AMMA and MMMA variants, both the AAM and AMMM variants formed mainly pentamers and hexamers. This observation accorded with that of Yoo et al. (38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) on human IgA1-human IgG2 domain swap mutants, which suggested that the Cα3 domain is required for IgA-like polymerization. In the same study, murine IgG2b-human IgM chimeras provided evidence that both Cμ3 and Cμ4 are needed for IgM-like polymerization (38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The fact that our AAM chimera formed mostly pentamers and hexamers, similar to IgM, demonstrated the validity of using IgA-IgM chimeras to identify domains responsible for the differential polymerization pattern of IgA and IgM. Thus, Cα2 or Cμ3, but not Cγ2 (38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), could support IgM-like polymerization. Taken together, our results pointed to the COOH-terminal domain as the main focus for further studies of isotype-specific polymerization motifs.J Chain Incorporation and Interactions with Free SC or pIgR of Polymeric ImmunoglobulinsOnly J chain-containing polymeric immunoglobulins can bind to the pIgR (5Johansen F.E. Braathen R. Brandtzaeg P. J. Immunol. 2001; 167: 5185-5192Crossref PubMed Scopus (166) Google Scholar, 11Brandtzaeg P. Prydz H. Nature. 1984; 311: 71-73Crossref PubMed Scopus (327) Google Scholar, 12Johansen F.E. Braathen R. Brandtzaeg P. Scand. J. Immunol. 2000; 52: 240-248Crossref PubMed Scopus (214) Google Scholar, 13Vaerman J.P. Langendries A. Giffroy D. Brandtzaeg P. Kobayashi K. Immunology. 1998; 95: 90-96Crossref PubMed Scopus (43) Google Scholar, 18Eskeland T. Brandtzaeg P. Immunochemistry. 1974; 11: 161-163Crossref PubMed Scopus (77) Google Scholar, 20Brandtzaeg P. Scand. J. Immunol. 1976; 5: 411-419Crossref PubMed Scopus (60) Google Scholar), and J chain-specific IgG antibodies or Fab fragments have been shown to inhibit binding of pIgA and pentameric IgM to free SC or the pIgR (10Brandtzaeg P. Scand. J. Immunol. 1975; 4: 837-842Crossref PubMed Google Scholar,14Vaerman J.P. Langendries A.E. Giffroy D.A. Kaetzel C.S. Fiani C.M. Moro I. Brandtzaeg P. Kobayashi K. Eur. J. Immunol. 1998; 28: 171-182Crossref PubMed Scopus (39) Google Scholar). In the present study, we found that the stoichiometry of J chain and λ light chain appeared to be similar for all recombinant immunoglobulin molecules except the AMMM chimera, which had a significantly reduced J chain content. Thus, J chain was abundantly present in pentamers from IgM as well as the AMMM and AAM variants but only at a low level in the AMMM variant. In IgA and the AMMA chimera, J chain was found in dimers and, especially with the AMMA chimera, also in larger polymers. Thus, isotype-specific J chain incorporation is determined by the COOH-terminal domain. Both Yoo et al. (38Yoo E.M. Coloma M.J. Trinh K.R. Nguyen T.Q. Vuong L.U. Morrison S.L. Chintalacharuvu K.R. J. Biol. Chem. 1999; 274: 33771-33777Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and we (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar) have previously found that chimeras of IgM and IgG need motifs from Cμ3 as well as Cμ4 for efficient J chain incorporation. Our present results suggested that Cα2 and Cμ3 are almost interchangeable in this respect. The role of the Cμ1 domain in J chain incorporation remains to be elucidated.In a recent study, one extra Cα3 domain from IgA2 was added onto IgG1, and the resulting polymers resembled pIgA in that they incorporated J chain and bound to the human pIgR (48Chintalacharuvu K.R. Vuong L.U. Loi L.A. Larrick J.W. Morrison S.L. Clin. Immunol. 2001; 101: 21-31Crossref PubMed Scopus (27) Google Scholar). It has been proposed that a predicted exposed loop of the Cα3 domain containing amino acids 402–410 (QEPSQGTTT), constitutes the pIgR binding site of pIgA (49Hexham J.M. White K.D. Carayannopoulos L.N. Mandecki W. Brisette R. Yang Y.S. Capra J.D. J. Exp. Med. 1999; 189: 747-752Crossref PubMed Scopus (53) Google Scholar). However, a naturally occurring mutant (protein 511) that lacks 36 amino acids in Cα3 (including amino acids 402–410) was found to complex with iodinated rat free SC and to be transported from blood into bile in a manner indistinguishable from pIgA (50Switzer I.C. Loney G.M. Yang D.S. Underdown B.J. Mol. Immunol. 1992; 29: 31-35Crossref PubMed Scopus (4) Google Scholar). To identify polymeric immunoglobulin domains involved in the pIgR binding sites, we exploited the fact that the rabbit receptor shows virtually no binding to human pentameric IgM but efficient binding to human pIgA, whereas human pIgR binds both ligands very efficiently. The recombinant rabbit-human chimeric free SC employed in our study (rD1-h SC) behaved like the rabbit receptor and was compared with the human counterpart. We found that all immunoglobulin variants bound quite well to human SC, except for the AMMM chimera, which had incorporated very little J chain. Both IgA and the AMMA chimera showed stronger binding to rD1-h SC than to human SC, in agreement with the higher level of binding shown by human pIgA for rabbit pIgR (28Røe M. Norderhaug I.N. Brandtzaeg P. Johansen F.E. J. Immunol. 1999; 162: 6046-6052PubMed Google Scholar). Our data suggested that elements in Cα3 are responsible for this efficient binding, which was supported by the fact that not only IgM but also the AAM chimera showed weaker binding to rabbit than to human SC. Our transcytosis experiments confirmed the functionality of the recombinant polymeric immunoglobulins by showing efficient transport of IgA and the AMMA chimera in MDCK cells transfected with either human pIgR, rabbit pIgR, or recombinant rD1-h pIgR. Also, IgM and the AAM chimera were transported by human pIgR but virtually not at all by rabbit pIgR or rD1-h pIgR. As expected, no receptor variant was able to transport the AMMM chimera, which had a low J chain content and low SC/pIgR binding capacity. Surprisingly, despite showing a very high level of binding to human free SC, the AAM chimera was not transported more efficiently than IgM by the human pIgR.The Cys-414 residues of Cμ3 may form disulfide bonds between the monomers in IgM (23Davis A.C. Roux K.H. Pursey J. Shulman M.J. EMBO J. 1989; 8: 2519-2526Crossref PubMed Scopus (86) Google Scholar), whereas the homologous Cys-309 of Cα2 is not involved in similar bonding between IgA subunits but instead forms a disulfide bridge to human SC (9Krugmann S. Pleass R.J. Atkin J.D. Woof J.M. J. Immunol. 1997; 159: 244-249PubMed Google Scholar, 27Fallgreen-Gebauer E. Gebauer W. Bastian A. Kratzin H.D. Eiffert H. Zimmermann B. Karas M. Hilschmann N. Biol. Chem. Hoppe. Seyler. 1993; 374: 1023-1028Crossref PubMed Scopus (57) Google Scholar). Interestingly, the AMMA chimera, which contained Cys-414 in Cμ3, also formed covalent complexes with human free SC. Thus, the Cys-414 residues were presumably not involved in intermolecular bonds in the AMMA dimer but were available for SC binding. In the AAM chimera, the Cα2 domain Cys-309 could apparently not make intermonomer bonds, because, similar to IgA, pentameric AAM formed covalent complexes with free SC (Fig. 4). The failure of such intramolecular disulfide stabilization may explain why the AAM variant was secreted in only very small amounts. Our result clearly showed that pentamer formation in itself does not restrict covalent SC binding. For IgM, we noted only a weak covalent association of SC with the pentamers, probably because most of the Cys-414 residues were engaged in intermonomer bonds in IgM and therefore unavailable for bridging to Cys-467 of human SC (23Davis A.C. Roux K.H. Pursey J. Shulman M.J. EMBO J. 1989; 8: 2519-2526Crossref PubMed Scopus (86) Google Scholar, 25Wiersma E.J. Shulman M.J. J. Immunol. 1995; 154: 5265-5272PubMed Google Scholar). It remains to be determined whether the covalent linking of SC to the AAM chimera made this pentamer more resistant to proteases, as has been shown for S-IgA (51Crottet P. Corthesy B. J. Immunol. 1998; 161: 5445-5453PubMed Google Scholar). It is also possible that the presence of Cα2 enhanced the noncovalent interactions between the AAM chimera and human SC because of progressive interactions between D2 and or D3 of human SC and Cα2 (52Norderhaug I.N. Johansen F.E. Krajci P. Brandtzaeg P. Eur. J. Immunol. 1999; 29: 3401-3409Crossref PubMed Scopus (36) Google Scholar).We have shown in this study, that the Cμ4 domain was sufficient for pentamer formation. Similarly, the Cα3 domain was sufficient for directing dimer formation. Cα3 also contained the unique motif for dimeric IgA to bind rabbit pIgR. Furthermore recombinant immunoglobulins containing Cμ4 failed to bind rabbit pIgR. We also produced a pentameric IgA-IgM chimeric variant that associated covalently with SC. Although such bonding did not increase ligand transport by human pIgR-transfected MDCK cells, it is known to stabilize S-IgA in secretions. At least 80% of all antibody-secreting plasma cells of the body are located in the gastrointestinal and respiratory mucosae, and most of them are committed to immunoglobulin A (IgA) production (1Brandtzaeg P. Farstad I.N. Johansen F.E. Morton H.C. Norderhaug I.N. Yamanaka T. Immunol. Rev. 1999; 171: 45-87Crossref PubMed Scopus (236) Google Scholar, 2Norderhaug I.N. Johansen F.E. Schjerven H. Brandtzaeg P. Crit. Rev. Immunol. 1999; 19: 481-508PubMed Google Scholar). All immunoglobulin isotypes consists of two heavy (H) and two light (L) chains, but for IgA, this H2L2 monomeric unit can polymerize further. Mucosally produced IgA consists predominantly of dimers and some larger polymers, collectively called polymeric IgA (pIgA).1 IgA polymerization is regulated by the incorporation of the joining chain (J chain) in that its presence greatly stimulates polymerization (3Sørensen V. Rasmussen I.B. Sundvold V. Michaelsen T.E. Sandlie I. Int. Immunol. 2000; 12: 19-27Crossref PubMed Scopus (53) Google Scholar, 4Vaerman J.P. Langendries A. Vander Maelen C. Immunol. Invest. 1995; 24: 631-641Crossref PubMed Scopus (35) Google Scholar, 5Johansen F.E. Braathen R. Brandtzaeg P. J. Immunol. 2001; 167: 5185-5192Crossref PubMed Scopus (166) Google Scholar, 6Johansen F.E. Natvig Norderhaug I. Røe M. Sandlie I. Brandtzaeg P. Eur. J. Immunol. 1999; 29: 1701-1708Crossref PubMed Scopus (42) Google Scholar, 7Ma J.K. Hiatt A. Hein M. Vine N.D. Wang F. Stabila P. van Dolleweerd C. Mostov K. Lehner T. Science. 1995; 268: 716-719Crossref PubMed Scopus (473) Google Scholar, 8Carayannopoulos L. Max E.E. Capra J.D. 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