X Wu1, M Kurpakus, L D Hazlett. 1. Department of Anatomy/Cell Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA.
Abstract
PURPOSE: The purpose of this study was to determine whether any of the previously identified human corneal epithelial pilus-binding proteins were cytokeratins. METHODS: Soluble human corneal epithelial proteins (hcep) were separated by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis under reducing conditions and transferred to nitrocellulose membrane for Western blot analysis. To characterize pilus-binding hcep (major proteins of < 21, 38, 45, 66 and 97 kDa and minor proteins, including a 55 kDa protein), blots were immunostained using three anti-keratin antibodies, including Pruss monoclonal antibody (MAb), specific for all classes of IFs, AE5 MAb, specific for a 64 kDa cytokeratin, and J7 polyclonal antibody (PAb), specific for a 55 kDa cytokeratin. In addition, major pilus-binding proteins were cut from 1-D SDS gel, electroeluted, dot blotted onto nitrocellulose membrane, and similarly analyzed. In addition, to further test whether any pilus-binding proteins were cytokeratins, soluble hcep were immunoprecipitated by MAb XLR-3, a specific anti-pilus antibody, after incubation with bacterial pili. The immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The blotted immunoprecipitated proteins were immunostained using Pruss MAb and MAb XLR-3. RESULTS: Immunoblots using Pruss MAb showed immunostaining of hcep of approximate molecular weights 45, 48, 55, 62 and 66 kDa. Other immunoblot analysis using AE5 MAb allowed identification of a 66 and a 45 kDa protein in both 1-D and dot blot analysis of eluted hcep. J7 PAb specifically immunostained a 55 kDa protein. In 2-D gel immunoblots, three 55 kDa proteins were immunostained by J7. Three proteins of molecular weights 45, 55 and 66 kDa, isolated after incubation of hcep and pili by immunoprecipitation with MAb XLR-3, also were positively immunostained by the Pruss MAb. CONCLUSIONS: Two of the previously identified major pilus-binding proteins of 45 and 66 kDa are cytokeratins.. Additionally, the 55 kDa minor pilus-binding protein is also a cytokeratin and appears to carry different electric charges. Novel approaches such as these provide new insight into the pathogenesis of P. aeruginosa infection in human cornea, and may lead to improved prevention and treatment of bacterial induced corneal disease.
PURPOSE: The purpose of this study was to determine whether any of the previously identified human corneal epithelial pilus-binding proteins were cytokeratins. METHODS: Soluble humancorneal epithelial proteins (hcep) were separated by one-dimensional (1-D) and two-dimensional (2-D) gel electrophoresis under reducing conditions and transferred to nitrocellulose membrane for Western blot analysis. To characterize pilus-binding hcep (major proteins of < 21, 38, 45, 66 and 97 kDa and minor proteins, including a 55 kDa protein), blots were immunostained using three anti-keratin antibodies, including Pruss monoclonal antibody (MAb), specific for all classes of IFs, AE5 MAb, specific for a 64 kDa cytokeratin, and J7 polyclonal antibody (PAb), specific for a 55 kDa cytokeratin. In addition, major pilus-binding proteins were cut from 1-D SDS gel, electroeluted, dot blotted onto nitrocellulose membrane, and similarly analyzed. In addition, to further test whether any pilus-binding proteins were cytokeratins, soluble hcep were immunoprecipitated by MAb XLR-3, a specific anti-pilus antibody, after incubation with bacterial pili. The immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The blotted immunoprecipitated proteins were immunostained using Pruss MAb and MAb XLR-3. RESULTS: Immunoblots using Pruss MAb showed immunostaining of hcep of approximate molecular weights 45, 48, 55, 62 and 66 kDa. Other immunoblot analysis using AE5 MAb allowed identification of a 66 and a 45 kDa protein in both 1-D and dot blot analysis of eluted hcep. J7 PAb specifically immunostained a 55 kDa protein. In 2-D gel immunoblots, three 55 kDa proteins were immunostained by J7. Three proteins of molecular weights 45, 55 and 66 kDa, isolated after incubation of hcep and pili by immunoprecipitation with MAb XLR-3, also were positively immunostained by the Pruss MAb. CONCLUSIONS: Two of the previously identified major pilus-binding proteins of 45 and 66 kDa are cytokeratins.. Additionally, the 55 kDa minor pilus-binding protein is also a cytokeratin and appears to carry different electric charges. Novel approaches such as these provide new insight into the pathogenesis of P. aeruginosa infection in human cornea, and may lead to improved prevention and treatment of bacterial induced corneal disease.