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Targeting mucosal sites by polymeric immunoglobulin receptor-directed peptides.

White KD, Capra JD - J. Exp. Med. (2002)

Bottom Line: Here by binding human secretory component to overlapping decapeptides of Calpha3, we confirm these residues and also uncover an additional site.Some transcytosis-selected peptides map to the same 402-410 pIgR-binding Calpha3 site.Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

ABSTRACT
Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402-410 of the Calpha3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Calpha3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Calpha3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402-410 pIgR-binding Calpha3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

Show MeSH
Transcytosis of GFP fusion proteins through nontransfected and pIgR-transfected MDCK cells. The native region from dIgA (amino acids 402–410) that was previously identified to be involved in pIgR-binding has been synthesized in both monomeric (mApep) and dimeric (dApep) form as GFP fusion proteins. The corresponding region from IgG that does not bind to the pIgR was also synthesized in monomeric form as a negative control (mGpep). The ability of the GFP fusion proteins to be specifically transported by the pIgR in the MDCK transcytosis system was assessed by measuring fluorescence from the apical medium after the MDCK transcytosis assay was performed using both nontransfected (shown as solid fill) and pIgR-transfected (shown as no fill) cells. Results represent means of three assays.
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fig2: Transcytosis of GFP fusion proteins through nontransfected and pIgR-transfected MDCK cells. The native region from dIgA (amino acids 402–410) that was previously identified to be involved in pIgR-binding has been synthesized in both monomeric (mApep) and dimeric (dApep) form as GFP fusion proteins. The corresponding region from IgG that does not bind to the pIgR was also synthesized in monomeric form as a negative control (mGpep). The ability of the GFP fusion proteins to be specifically transported by the pIgR in the MDCK transcytosis system was assessed by measuring fluorescence from the apical medium after the MDCK transcytosis assay was performed using both nontransfected (shown as solid fill) and pIgR-transfected (shown as no fill) cells. Results represent means of three assays.

Mentions: To identify a motif on IgA dimer molecules or unique peptide motifs that lead to secretion onto mucosal surfaces via the pIgR-mediated transcytosis pathway, experiments were designed to localize the smallest possible peptide(s) required for the pIgR–dIgA interaction. The native region from IgA (amino acids 402–410) that we have shown previously to be involved in pIgR-binding was synthesized in both monomeric (mApep) and dimeric (dApep) form with a small linker as GFP fusion proteins. As a negative control, the corresponding region from Cγ3 (amino acids 398–403) was also synthesized as a fusion protein in monomeric form (mGpep). The ability of mApep, dApep, and mGpep to be specifically transported by the pIgR was assessed in the MDCK transcytosis assay by using both pIgR-transfected and nontransfected cells (Fig. 2) . The data indicate that a peptide derived from the native region from the CH3 region of IgA in dimeric form, and to a lesser extent in monomeric form, can indeed direct transport of a fusion protein through pIgR-transfected MDCK cells but not through nontransfected MDCK cells. mGpep, the negative control peptide derived from the sequence of IgG, was not transported by the pIgR.


Targeting mucosal sites by polymeric immunoglobulin receptor-directed peptides.

White KD, Capra JD - J. Exp. Med. (2002)

Transcytosis of GFP fusion proteins through nontransfected and pIgR-transfected MDCK cells. The native region from dIgA (amino acids 402–410) that was previously identified to be involved in pIgR-binding has been synthesized in both monomeric (mApep) and dimeric (dApep) form as GFP fusion proteins. The corresponding region from IgG that does not bind to the pIgR was also synthesized in monomeric form as a negative control (mGpep). The ability of the GFP fusion proteins to be specifically transported by the pIgR in the MDCK transcytosis system was assessed by measuring fluorescence from the apical medium after the MDCK transcytosis assay was performed using both nontransfected (shown as solid fill) and pIgR-transfected (shown as no fill) cells. Results represent means of three assays.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196054&req=5

fig2: Transcytosis of GFP fusion proteins through nontransfected and pIgR-transfected MDCK cells. The native region from dIgA (amino acids 402–410) that was previously identified to be involved in pIgR-binding has been synthesized in both monomeric (mApep) and dimeric (dApep) form as GFP fusion proteins. The corresponding region from IgG that does not bind to the pIgR was also synthesized in monomeric form as a negative control (mGpep). The ability of the GFP fusion proteins to be specifically transported by the pIgR in the MDCK transcytosis system was assessed by measuring fluorescence from the apical medium after the MDCK transcytosis assay was performed using both nontransfected (shown as solid fill) and pIgR-transfected (shown as no fill) cells. Results represent means of three assays.
Mentions: To identify a motif on IgA dimer molecules or unique peptide motifs that lead to secretion onto mucosal surfaces via the pIgR-mediated transcytosis pathway, experiments were designed to localize the smallest possible peptide(s) required for the pIgR–dIgA interaction. The native region from IgA (amino acids 402–410) that we have shown previously to be involved in pIgR-binding was synthesized in both monomeric (mApep) and dimeric (dApep) form with a small linker as GFP fusion proteins. As a negative control, the corresponding region from Cγ3 (amino acids 398–403) was also synthesized as a fusion protein in monomeric form (mGpep). The ability of mApep, dApep, and mGpep to be specifically transported by the pIgR was assessed in the MDCK transcytosis assay by using both pIgR-transfected and nontransfected cells (Fig. 2) . The data indicate that a peptide derived from the native region from the CH3 region of IgA in dimeric form, and to a lesser extent in monomeric form, can indeed direct transport of a fusion protein through pIgR-transfected MDCK cells but not through nontransfected MDCK cells. mGpep, the negative control peptide derived from the sequence of IgG, was not transported by the pIgR.

Bottom Line: Here by binding human secretory component to overlapping decapeptides of Calpha3, we confirm these residues and also uncover an additional site.Some transcytosis-selected peptides map to the same 402-410 pIgR-binding Calpha3 site.Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunogenetics Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA.

ABSTRACT
Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402-410 of the Calpha3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Calpha3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Calpha3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402-410 pIgR-binding Calpha3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

Show MeSH