Characterization of P. aeruginosa pili binding human corneal epithelial proteins.
Soluble human corneal epithelial proteins (hcep) to which P. aeruginosa pili bind were identified and characterized using gel electrophoresis and Western blotting techniques. Pilus binding proteins were identified using nitrocellulose membrane blots of one dimensional polyacrylamide gels (1-D-SDS-PAGE) of solubilized hcep and a pilus overlay assay. Five major proteins of approximate molecular weights < 21, 38, 45, 66, 97 (a doublet) kilodaltons (kDa) and two additional proteins of > 97 kDa bound pili using an overlay assay and immunoblotting with the monoclonal antibody (MAb) XLR-3, specific for pili. Several of these pili binding proteins were confirmed as single proteins using a similar pilus overlay assay and blotted proteins from two-dimensional polyacrylamide gels (2-D-SDS-PAGE) of hcep. A solid-phase binding assay confirmed that pili binding to hcep was specific, competitive and saturable. The importance of the carbohydrate portions of corneal pili binding proteins was assessed by preincubation of 1-D gel blots of hcep or dot blots of selected eluted pilus binding proteins ( < 21, 38, 45, 66 and 97 kDa) with periodic acid. Mild periodate oxidation of blots before pilus overlay assay completely abolished pili binding. The role of glycosylation of proteins also was assessed using 1-D blots of hcep or dot blots of eluted pilus binding proteins. These blots were preincubated with different lectins before incubation with pili in the pilus overlay assay. Of several lectins examined, only sConA, which recognizes terminal mannose residues, prevented pili binding in the pilus overlay assay. These studies provide evidence that several human corneal epithelial glycosylated proteins provide receptor sites for bacterial pili binding, and that the binding of pili to these proteins is specific, competitive and saturable. They also show that the carbohydrate mannose functions as an integral component of hcep pili binding receptors.
Department of Anatomy/Cell Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
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Wu
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Gupta
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Hazlett
D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001710:Biotin; D015153:Blotting, Western; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037102:Lectins; D008358:Mannose / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000378:metabolism* / Q000648:ultrastructure; D011971:Receptors, Immunologic / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005136:Eye Proteins; D006023:Glycoproteins; D037102:Lectins; D011971:Receptors, Immunologic; C037872:pili, bacterial receptor; D001710:Biotin; D008358:Mannose