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A human immunoglobulin (Ig)A calpha3 domain motif directs polymeric Ig receptor-mediated secretion.

Hexham JM, White KD, Carayannopoulos LN, Mandecki W, Brisette R, Yang YS, Capra JD - J. Exp. Med. (1999)

Bottom Line: Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a beta-strand immediately NH2-terminal to this loop had no effect.These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Calpha3 domain, which directs mucosal secretion of polymeric antibodies.We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.

ABSTRACT
Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a panel of human IgA1/IgG1 constant region "domain swap" mutants, the binding site for the pIgR on dimeric IgA (dIgA) was localized to the Calpha3 domain. Selection of random peptides for pIgR binding and comparison with the IgA sequence suggested amino acids 402-410 (QEPSQGTTT), in a predicted exposed loop of the Calpha3 domain, as a potential binding site. Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a beta-strand immediately NH2-terminal to this loop had no effect. All pIgR binding IgA sequences contain a conserved three amino acid insertion, not present in IgG, at this position. These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Calpha3 domain, which directs mucosal secretion of polymeric antibodies. We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.

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Alignment of deduced peptide  sequences from selection of phage display peptide library against pIgR receptor–expressing  cells with the human Cα3 domain amino  acid sequence. Peptides designated A or M  are from the acid-eluted and cell-associated  fractions, respectively. Numbering of IgA1  is according to reference 5.
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Figure 2: Alignment of deduced peptide sequences from selection of phage display peptide library against pIgR receptor–expressing cells with the human Cα3 domain amino acid sequence. Peptides designated A or M are from the acid-eluted and cell-associated fractions, respectively. Numbering of IgA1 is according to reference 5.

Mentions: dIgA contains four Cα3 domains and the covalently bound J chain which, together with the IgA tailpiece, are responsible for IgA polymerization. To reduce the complexity of this problem, a library of random 40-mer peptides, expressed as a phage display library (20), was selected against pIgR-expressing MDCK cells. The goal was to identify putative pIgR binding sites within IgA by reducing them to a minimum peptide binding unit, a proven approach for several receptor–ligand interactions (26–28). Selection was carried out on live pIgR-expressing MDCK cells in suspension with negative selection on nonreceptor– expressing cells. Bound phage were eluted with acid or by cell lysis. Recovery of both acid-eluted and cell-associated phage increased gradually from ∼6 × 104 to 5 × 107 CFU over 4–6 successive rounds, indicating enrichment for specific binding clones. Individual clones were randomly selected from the final panning from the acid-eluted and membrane-associated fractions and sequenced. Binding of the enriched phage populations to recombinant human pIgR, as measured by ELISA, increased with successive rounds of panning and was inhibited by polymeric IgM (data not shown). Sequencing of phagemid DNA showed that 20 out of 32 acid-eluted clones and 12 out of 32 cell-associated clones had open reading frames (Fig. 2). There is little clonality among these two groups of sequences, although the A22 peptide was recovered three times. These peptides were aligned for maximum homology with the human IgA1 Cα3 region amino acid sequence (Fig. 2) using the PIMA program (23). Many of the peptides, particularly A12 (9 out of 30 identical amino acids) (Fig. 3 a), show homology with human IgA1 Cα3 domain, prompting a further examination of the amino acid sequence and structure in this area.


A human immunoglobulin (Ig)A calpha3 domain motif directs polymeric Ig receptor-mediated secretion.

Hexham JM, White KD, Carayannopoulos LN, Mandecki W, Brisette R, Yang YS, Capra JD - J. Exp. Med. (1999)

Alignment of deduced peptide  sequences from selection of phage display peptide library against pIgR receptor–expressing  cells with the human Cα3 domain amino  acid sequence. Peptides designated A or M  are from the acid-eluted and cell-associated  fractions, respectively. Numbering of IgA1  is according to reference 5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192928&req=5

Figure 2: Alignment of deduced peptide sequences from selection of phage display peptide library against pIgR receptor–expressing cells with the human Cα3 domain amino acid sequence. Peptides designated A or M are from the acid-eluted and cell-associated fractions, respectively. Numbering of IgA1 is according to reference 5.
Mentions: dIgA contains four Cα3 domains and the covalently bound J chain which, together with the IgA tailpiece, are responsible for IgA polymerization. To reduce the complexity of this problem, a library of random 40-mer peptides, expressed as a phage display library (20), was selected against pIgR-expressing MDCK cells. The goal was to identify putative pIgR binding sites within IgA by reducing them to a minimum peptide binding unit, a proven approach for several receptor–ligand interactions (26–28). Selection was carried out on live pIgR-expressing MDCK cells in suspension with negative selection on nonreceptor– expressing cells. Bound phage were eluted with acid or by cell lysis. Recovery of both acid-eluted and cell-associated phage increased gradually from ∼6 × 104 to 5 × 107 CFU over 4–6 successive rounds, indicating enrichment for specific binding clones. Individual clones were randomly selected from the final panning from the acid-eluted and membrane-associated fractions and sequenced. Binding of the enriched phage populations to recombinant human pIgR, as measured by ELISA, increased with successive rounds of panning and was inhibited by polymeric IgM (data not shown). Sequencing of phagemid DNA showed that 20 out of 32 acid-eluted clones and 12 out of 32 cell-associated clones had open reading frames (Fig. 2). There is little clonality among these two groups of sequences, although the A22 peptide was recovered three times. These peptides were aligned for maximum homology with the human IgA1 Cα3 region amino acid sequence (Fig. 2) using the PIMA program (23). Many of the peptides, particularly A12 (9 out of 30 identical amino acids) (Fig. 3 a), show homology with human IgA1 Cα3 domain, prompting a further examination of the amino acid sequence and structure in this area.

Bottom Line: Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a beta-strand immediately NH2-terminal to this loop had no effect.These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Calpha3 domain, which directs mucosal secretion of polymeric antibodies.We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.

ABSTRACT
Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a panel of human IgA1/IgG1 constant region "domain swap" mutants, the binding site for the pIgR on dimeric IgA (dIgA) was localized to the Calpha3 domain. Selection of random peptides for pIgR binding and comparison with the IgA sequence suggested amino acids 402-410 (QEPSQGTTT), in a predicted exposed loop of the Calpha3 domain, as a potential binding site. Alanine substitution of two groups of amino acids in this area abrogated the binding of dIgA to pIgR, whereas adjacent substitutions in a beta-strand immediately NH2-terminal to this loop had no effect. All pIgR binding IgA sequences contain a conserved three amino acid insertion, not present in IgG, at this position. These data localize the pIgR binding site on dimeric human IgA to this loop structure in the Calpha3 domain, which directs mucosal secretion of polymeric antibodies. We propose that it may be possible to use a pIgR binding motif to deliver antigen-specific dIgA and small-molecule drugs to mucosal epithelia for therapy.

Show MeSH