Two corneal diseases will be investigated: Macular corneal dystrophy: Abnormal glycoconjugates synthesized by corneas with macular dystrophy in organ culture will be characterized to establish the cause of the abnormal lectin binding pattern known to be present in corneas with macular dystrophy. In addition we will attempt to determine whether the defect in sulfation of keratan sulfate (KS) proteoglycan in corneas with macular dystrophy is due to the deficiency of KS-sulfotransferases in the diseased corneas. This study may enable us to elucidate the biochemical abnormalities responsible for the development of macular corneal dystrophy. Keratoconus: We have shown by 2D-electrophoresis that, compared to normal corneas, a number of additional proteins (K) are present and a number of normal corneal proteins (N) are absent in keratoconus corneas. The proposed study will attempt to determine whether the """"""""K"""""""" proteins in keratoconus have been derived from the """"""""N"""""""" proteins. Polyclonal antibodies will be prepared against one of the """"""""N"""""""" proteins. These antibodies will be used to: (1) stain the Western blots of 2D-gels of proteins from normal and keratoconus corneas to determine whether one or more of the """"""""K"""""""" proteins are related to the """"""""N"""""""" protein and (2) study the synthesis of """"""""N"""""""" and """"""""K"""""""" proteins by corneas in organ culture by immunoprecipitation to determine whether one or more """"""""K"""""""" proteins in keratoconus appears as a consequence of the abnormal processing of the normal corneal protein (N). The cDNA libraries of normal and keratoconus corneas will be prepared in lambda gtll expression vector and the nucleotide sequence of full-length cDNA inserts of the """"""""N"""""""" protein and its abnormal counterpart (K) in keratoconus, if found, will be determined. Amino acid sequence of the """"""""N"""""""" and """"""""K"""""""" proteins will be analyzed for homology with the sequences in the databases of the National Biomedical Research Foundation. We anticipate that this study will help us identify and characterize the proteins involved in the development of keratoconus. (See rebuttal to the previous summary statement, pages 20-22.)
