specific chimeric iga1   and iga1/igg1 domain swap mutants were expressed as previously described ( 16 , 17 ) . antibodies were eluted with 200 mm arsanillic acid ( sigma chemical co. ) in 200 mm tris - hcl , ph 8.0 ,   which was removed by extensive dialysis against pbs . mono-   and dimeric iga were detected by 4% nonreducing sds - page   analysis . the hexahistidine - tagged human pigr extracellular domain was expressed in a similar manner and purified on a ni - nta agarose ( qiagen ) column ( 18 ) . the c3 loop mutants l1 , l2 , and l3 were constructed by pcr   soeing ( splicing by overlap extension ; reference 19 ) using the   following complementary pairs of sense ( s ) and antisense ( as )   primers : l1s 5-gagcccagcgcgggcgccgccgccttcgctgtg-3 , l1as 5-ctcgggtcgcgcccgcggcggc - ggaagcgacac-3 ; l2s 5-tacctgactgcggcagccgcgcaggagccc-3 , l2as 5-atggactgacgccgtc - ggcgcgtcctcggg-3 ; and l3s 5-cggcaggaggccgccgcggccaccaccacc-3 , l3as 5-gccgtcctccggcggcgccggtggtggtgg-3. the outer primers   b1 - 2 ( 5-cctataaccatgggatggagcttcatc-3 ) , specific for the 5 leader of the vh region of this chimeric iga heavy   chain , and c3 - 3 ( 5-ccctctagattagtagcaggtgccgtccac-3 ) , specific for the 3 tailpiece  encoding sequence   of the iga1 gene , were used with the above primer pairs l1l3   to generate pairs of 5 and 3 fragments with complementary   overlaps . these fragments were gel purified then spliced in a further pcr reaction using the outer primers b1 - 2 and c3 - 3.   modified iga1 genes were cloned into the baculovirus transfer   vector using xbai and ncoi digestion and the insert sequences   were verified . madin - derby canine kidney ( mdck ) cells   were placed in serum - free mem plus earle 's salts ( mediatech ,   inc . ) cells were harvested in 10 mm   edta in pbs and washed in pbs 0.1% bsa . pigr binding of   human iga1 antibodies and mutants was assessed by incubation   of 100 l antibody in pbs / bsa with 10 cells for 1 h. cells   were washed three times in pbs / bsa and bound antibody was   detected with 100 l of an anti  human  fitc conjugate ( sigma   chemical co. ) diluted 1:100 . data collection and   analysis were performed with the lysysii ( becton dickinson )   and winmidi ( http://facs.scripps.edu ) or with the cellquest   programs ( becton dickinson ) . the random 40-mer   peptide library was constructed in the pcantab5e vector and   has an actual total diversity of 1.55  10 ( 20 ) . the random 40-mer is flanked by two peptide tag sequences , preceded by a leader   peptide and fused to the membrane - proximal domain of the m13   phage coat protein iii . 12  10 mdck cells were harvested in   5 ml pbs plus 10 mm edta at 37c , washed twice in 15 ml   pbs , and resuspended in 1.8 ml pbs at 4c . 100 l phagemid library stock ( 4.5  10 cfu ) was added and incubated for 1 h at   37 or 4c . bound phage were eluted with 2 ml of 0.1 m glycine/ hcl , ph 2.2 , containing 0.1% bsa for 10 min and neutralized   immediately with 400 l of 2 m tris base . phage rescue and amplification were carried out in escherichia coli strain tg1 ( pharmacia ) according to standard procedures ( 21 ) . dna sequencing was carried   out on double - stranded plasmid or phagemid dna using an abi   377 prism ( applied biosystems , inc . ) alignments of deduced peptide sequences and ig - constant regions   were carried out using the map ( 22 ) and pima ( 23 ) software . specific chimeric iga1   and iga1/igg1 domain swap mutants were expressed as previously described ( 16 , 17 ) . antibodies were eluted with 200 mm arsanillic acid ( sigma chemical co. ) in 200 mm tris - hcl , ph 8.0 ,   which was removed by extensive dialysis against pbs . mono-   and dimeric iga were detected by 4% nonreducing sds - page   analysis . the hexahistidine - tagged human pigr extracellular domain was expressed in a similar manner and purified on a ni - nta agarose ( qiagen ) column ( 18 ) . the c3 loop mutants l1 , l2 , and l3 were constructed by pcr   soeing ( splicing by overlap extension ; reference 19 ) using the   following complementary pairs of sense ( s ) and antisense ( as )   primers : l1s 5-gagcccagcgcgggcgccgccgccttcgctgtg-3 , l1as 5-ctcgggtcgcgcccgcggcggc - ggaagcgacac-3 ; l2s 5-tacctgactgcggcagccgcgcaggagccc-3 , l2as 5-atggactgacgccgtc - ggcgcgtcctcggg-3 ; and l3s 5-cggcaggaggccgccgcggccaccaccacc-3 , l3as 5-gccgtcctccggcggcgccggtggtggtgg-3. the outer primers   b1 - 2 ( 5-cctataaccatgggatggagcttcatc-3 ) , specific for the 5 leader of the vh region of this chimeric iga heavy   chain , and c3 - 3 ( 5-ccctctagattagtagcaggtgccgtccac-3 ) , specific for the 3 tailpiece  encoding sequence   of the iga1 gene , were used with the above primer pairs l1l3   to generate pairs of 5 and 3 fragments with complementary   overlaps . these fragments were gel purified then spliced in a further pcr reaction using the outer primers b1 - 2 and c3 - 3.   modified iga1 genes were cloned into the baculovirus transfer   vector using xbai and ncoi digestion and the insert sequences   were verified . madin - derby canine kidney ( mdck ) cells   were placed in serum - free mem plus earle 's salts ( mediatech ,   inc . ) cells were harvested in 10 mm   edta in pbs and washed in pbs 0.1% bsa . pigr binding of   human iga1 antibodies and mutants was assessed by incubation   of 100 l antibody in pbs / bsa with 10 cells for 1 h. cells   were washed three times in pbs / bsa and bound antibody was   detected with 100 l of an anti  human  fitc conjugate ( sigma   chemical co. ) diluted 1:100 . data collection and   analysis were performed with the lysysii ( becton dickinson )   and winmidi ( http://facs.scripps.edu ) or with the cellquest   programs ( becton dickinson ) . the random 40-mer   peptide library was constructed in the pcantab5e vector and   has an actual total diversity of 1.55  10 ( 20 ) . the random 40-mer is flanked by two peptide tag sequences , preceded by a leader   peptide and fused to the membrane - proximal domain of the m13   phage coat protein iii . 12  10 mdck cells were harvested in   5 ml pbs plus 10 mm edta at 37c , washed twice in 15 ml   pbs , and resuspended in 1.8 ml pbs at 4c . 100 l phagemid library stock ( 4.5  10 cfu ) was added and incubated for 1 h at   37 or 4c . bound phage were eluted with 2 ml of 0.1 m glycine/ hcl , ph 2.2 , containing 0.1% bsa for 10 min and neutralized   immediately with 400 l of 2 m tris base . phage rescue and amplification were carried out in escherichia coli strain tg1 ( pharmacia ) according to standard procedures ( 21 ) . dna sequencing was carried   out on double - stranded plasmid or phagemid dna using an abi   377 prism ( applied biosystems , inc . ) automated sequencer . alignments of deduced peptide sequences and ig - constant regions   were carried out using the map ( 22 ) and pima ( 23 ) software . chimeric human iga1 ( 16 ) and a panel of iga1/igg1   constant region domain swap mutants ( 24 ) with murine - encoded arsonate specificity were expressed in baculovirus   as both monomer and dimer , affinity purified , and used to   define the pigr binding site . diga was operationally defined as an iga preparation generated by coexpression of   iga with j chain . mdck cells , transfected with rabbit   pigr ( 25 ) , were used to measure binding of recombinant   iga1 mutants to the receptor by facs analysis ( fig . mutant vgaa , in which the c1 domain was substituted with the c1 domain , bound to the   pigr in a manner similar to wild - type iga1 ( fig . . 1 c , heavy line ) bound to the receptor , whereas the monomer ( light line ) did not . similarly ,   the vgga mutant , in which both c1 and c2 including   the hinge of iga were replaced with the analogous domains   from igg , bound as a dimer but not as a monomer ( fig . thus , the c1 and c2 domains of diga are not necessary for pigr binding , suggesting that the presence of the   c3 domain is required . diga contains four c3 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 ( 2628 ) . selection was carried out on live pigr - expressing mdck   cells in suspension with negative selection on nonreceptor expressing cells . recovery of both acid - eluted and cell - associated   phage increased gradually from 6  10 to 5  10 cfu   over 46 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 . 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 c3 region amino acid sequence ( fig . many of the peptides , particularly a12 ( 9 out of 30 identical amino acids ) ( fig . 3 a ) ,   show homology with human iga1 c3 domain , prompting a further examination of the amino acid sequence and   structure in this area . the human c3 domain is 40% identical and 62% homologous to the corresponding region of human igg1 at   the amino acid level . accordingly , the human igg1 crystal structure ( 29 )   was used to predict the likely positions of the major structural motifs ( -strands and loops ) within the iga1 sequence , an approach used previously to map the fcr   ( cd89 ) binding site on iga1 ( 24 ) . 3 a shows the alignment of the peptide a12 with the iga1 sequence and the   corresponding igg1 sequence with its secondary structural   features . the a12 peptide is homologous to a region that   in the igg structure forms an exposed 6-amino acid loop   between two -strands . however , in iga1 , this area contains   a 3-amino acid insertion to expand the loop to 9-amino   acids . the flanking -strand sequences and part of the loop   are conserved between iga and igg , which suggests that   gross structural features are also conserved . 3 b shows   alignment of this region in the ch3 domain of five mammalian iga molecules aligned with the four human igg   subclasses . despite sequence differences in the loop , all iga   sequences have the three additional amino acids , whereas   the igg sequences do not . similar to iga , the sequence of   igm contains a 2-amino acid insertion at this site ( data not   shown ) . on the basis of these observations , three mutant   iga1 molecules were constructed and expressed in baculovirus to examine the effect of amino acid changes in this   area on pigr binding ( fig . mutations were made in   the loop itself ( l1 and l3 ) and in the -strand nh2-terminal to the loop ( l2 ) as a negative control . binding was then   measured to the physiologically relevant human receptor   by elisa using the purified recombinant extracellular domain of human pigr expressed in baculovirus as previously   described ( 18 ) . 4 shows the binding of iga1 monomer ,   iga1 dimer , and igg compared with the monomeric and   dimeric forms of the l1 , l2 , and l3 mutants to purified   human pigr . only dimeric wild - type iga1 and dimeric l2   mutant , in which the mutations are in the -strand nh2-terminal to the loop , mutations within the   loop itself , namely l1 and l3 , abrogate the binding of the   dimeric iga1 mutant molecules to the pigr . similar binding patterns were obtained with the loop mutants and rabbit pigr - expressing cells as measured by facs ( data not   shown ) . these results indicate that this c3 loop is the major binding motif for the pigr on diga . iga is , in functional terms , closely related to igm , sharing its ability to polymerize and be secreted . the presence of amino acid sequence insertions in all the   polymeric igs that are ligands for this receptor and the absence of insertions from non - pigr binding igs ( fig . the variation in the insertion size and the actual iga and igm sequences may reflect   differences in fine structure of these polymeric antibodies   or in their affinity for pigr binding . the fact that monomeric iga is not secreted suggests   that either a conformational change induced by polymerization is required for diga binding to the receptor or that   the binding requires a polyvalent interaction of these c3   sites with the receptor . the presence of j chain is required   for optimal iga ( or igm ) polymerization but its precise role   in ig secretion remains to be elucidated . the increase in   binding observed with dimeric l3 when compared with   monomeric l3 ( and to a lesser extent with the l1 mutants )   suggests that j chain and/or polymerization may play a role   in binding ( fig . although amino acids 402410 in the   c3 domain of diga define a major pigr binding site ,   other diga structures may be involved . deficient   mice express lower levels of polymeric iga and have impaired hepatic transport of iga ( which humans lack ) but   normal levels of iga at mucosal epithelial sites , compared   with wild - type mice ( 30 , 31 ) . j chain thus may not be necessary for secretion of iga but still required for stable binding to the secretory component in the mucosal environment ; however , alternative secretory mechanisms may also   be involved . further studies are underway with peptides   and additional mutations to examine the nature of the interaction between iga and the pigr as well as the role of   j chain . the ability of a peptide sequence to confer mucosal   secretion upon a molecule may prove a powerful means of   delivery of therapeutic molecules to mucosal areas where   they may prevent the entry of pathogens . binding of monomeric and dimeric iga / igg domain swap   mutant antibodies to pigr expressed on mdck cells . ( a ) staining of   mdck cells with sheep anti - pigr ( heavy line ) antiserum or normal   sheep serum ( broken line ) followed by anti - sheep igg fitc conjugate . ( b ) binding of wild - type iga monomer ( thin line ) or dimer ( heavy line )   to pigr on mdck cells . ( c ) binding of vgaa mutant expressed as   monomer ( thin line ) or dimer ( heavy line ) to pigr on mdck cells . ( d )   binding of vgga mutant expressed as monomer ( thin line ) or dimer   ( heavy line ) to pigr on mdck cells . alignment of deduced peptide   sequences from selection of phage display peptide library against pigr receptor  expressing   cells with the human c3 domain amino   acid sequence . peptides designated a or m   are from the acid - eluted and cell - associated   fractions , respectively . comparison of igg1 and iga1 ch3 sequences and igg1   structure in the area homologous to several phage - derived peptides . iggstr indicates structural features of igg1 where < denotes a -strand running in a descending   orientation ( i.e. , hinge to ch3 direction ) , > denotes a -strand running in   an ascending direction ( i.e. , ch3 to hinge direction ) , and  denotes a loop or   open structure ( 29 ) . ( b ) comparison of several mammalian iga sequences   with the four human igg subclasses showing the additional iga - specific   amino acids present in the loop at positions 402410 in the iga sequence . hu , human ; gr , gorilla ; mur , murine ; rab , rabbit . ( c ) iga1 c3 mutants l1 ,   l2 , and l3 aligned with the c3 and c3 wild - type sequences and c3   structure ( iggstr ) . = denotes sequence identity in the mutants ,  denotes   a space introduced in the igg sequence to maximize homology , and   iggstr is labeled according to panel a. numbering of iga1 and igg1 is   according to references 5 and 29 , respectively . binding of iga mutants l1 , l2 , and l3 to purified   human pigr by elisa . the extracellular domain of human   pigr was purified after expression in baculovirus and coated   onto elisa plates at 10 g / ml .   chimeric iga1 and iga1 c3   mutants l1 , l2 , and l3 were expressed as both monomeric ( m )   and dimeric ( d ) forms along with chimeric igg1 , purified and incubated   on the pigr - coated plates to compare their abilities to bind to pigr .
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 c3 domain . 
 selection of random peptides   for pigr binding and comparison with the iga sequence suggested amino acids 402410   ( qepsqgttt ) , in a predicted exposed loop of the c3 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 -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 c3 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 .