Xa. Wu et al., CHARACTERIZATION OF PSEUDOMONAS-AERUGINOSA PILI BINDING HUMAN CORNEALEPITHELIAL PROTEINS, Current eye research, 14(10), 1995, pp. 969-977
Soluble human corneal epithelial proteins (hcep) to which P. aeruginos
a pili bind were identified and characterized using gel electrophoresi
s and Western blotting techniques. Pilus binding proteins were identif
ied using nitrocellulose membrane blots of one dimensional polyacrylam
ide 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 k
Da bound pili using an overlay assay and immunoblotting with the monoc
lonal antibody (MAb) XLR-3, specific for pili. Several of these pili b
inding proteins were confirmed as single proteins using a similar pilu
s overlay assay and blotted proteins from two-dimensional polyacrylami
de 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 prote
ins was assessed by preincubation of 1-D gel blots of hcep or dot blot
s of selected eluted pilus binding proteins (<21, 38, 45, 66 and 97 kD
a) with periodic acid. Mild periodate oxidation of blots before pilus
overlay assay completely abolished pili binding. The role of glycosyla
tion of proteins also was assessed using 1-D blots of hcep or dot blot
s of eluted pilus binding proteins. These blots were preincubated with
different lectins before incubation with pili in the pilus overlay as
say. Of several lectins examined, only sCon A, which recognizes termin
al 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 bindi
ng, and that the binding of pili to these proteins is specific, compet
itive and saturable. They also show that the carbohydrate mannose func
tions as an integral component of hcep pili binding receptors.