The presence of keratin 5 as an IgG Fc binding protein in human corneal epithelium

Abstract

Rabbit immunoglobulin G (IgG) binds specifically to the human corneal epithelium. We investigated this phenomenon to identify the binding material in the tissue. Sections of human cornea were immunostained with negative-control grade rabbit IgG, F(ab′) 2 (the antigen recognition fragment of IgG yielded by pepsin digestion), and Fc fragments. Human corneal epithelium was homogenized in a buffer containing Triton X-100 and the water-insoluble residue was subjected to two-dimensional gel electrophoresis. Western blotting was performed on the gels to detect materials that bound with rabbit IgG Fc fragments. Spots in the gel that bound with the Fc fragments were collected and analysed by peptide mass fingerprinting (PMF). Using an antibody against a binding candidate, keratin 5 (suggested by PMF analysis), we checked the occurrence of this phenomenon. Immunohistochemistry showed that rabbit IgG bound to corneal epithelium via the Fc region but not F(ab′) 2. Western blots using rabbit Fc fragments showed that some protein spots in the gel bound to the fragments. All of the binding materials were basic and had molecular weights of approximately 55 kDa. PMF analysis showed that the binding material was keratin 5; all binding materials on the transferred membrane were recognized by an anti-keratin 5 antibody. On corneal sections, anti-keratin 5 produced staining similar to that of rabbit IgG. These findings suggest that rabbit IgG binds to keratin 5 in the human corneal epithelium via the Fc region of the molecule.