Structural constraints for the binding of short peptides to claudin-4 revealed by surface plasmon resonance.
Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: <XX(Y/W)(X)(3 or 4)Y(Y/X)(L/I)XX>. Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins.
Ling
Jun
J
Division of Biomedical Sciences, University of California, Riverside, California, USA.
Liao
Hailing
H
Clark
Robin
R
Wong
Mandy Sze Man
MS
Lo
David D
DD
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D062506:Claudin-4; D003016:Clostridium perfringens / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006389:Hemagglutinins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods
C568157:CLDN4 protein, human; D062506:Claudin-4; C568158:Cldn4 protein, mouse; D004731:Endotoxins; D006389:Hemagglutinins, Viral; D008565:Membrane Proteins; D010455:Peptides; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; C048071:endotoxin receptor
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