Identification and characterization of the OCRL (Lowe`s Syndrome) protein

The oculocerebrorenal syndrome of Lowe is a rare X-linked disorder characterized by congenital cataracts, proximal renal tubular dysfunction, and mental retardation. The OCRL gene, OCRL-1, was identified by positional cloning and found to be highly homologous to a 75 kDa inositol polyphosphate 5-phosphatase (INPP5B). GTPase activating proteins from the rho-GAP family have a lower degree of homology to OCRL-1. These similarities suggest a role for the OCRL protein in a signalling pathway and particularly in the inositol pathway. To begin characterization of the OCRL protein, antibodies were made to identify the protein. The carboxy and amino terminal portions of OCRL-1 were expressed in E. coli and used as antigens to raise polyclonal antisera. These were affinity purified against their respective antigen bound to PVDF membranes. A single protein of 105 kDa was identified using both affinity purified antisera in Western blots of normal fibroblasts and murine embryonal stem (ES) cell lines. This protein was absent in the fibroblasts of a Lowe`s syndrome patient who lacked OCRL-1 message and in ES cells carrying a major disruption of the OCRL-1 gene. Affinity purified antisera recognize a protein of similar size in many mouse tissues and in the yeast, S. cerevisiae. Initial studiesmore » of fibroblasts from Lowe`s syndrome patients by Western analysis support the RNA expression data and reveal normal and abnormal sizes and expression levels of the OCRL protein. Immunohistochemical studies will be used to localize the protein in cells and in various organs. Initial characterization of the OCRL protein will elucidate its likely role in signal transduction and/or inositol metabolism.« less