Immunological identification, localization and characterization of the Lowe syndrome (OCRL) protein

Abstract

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 Kd inositol polyphosphate 5-phosphatase, INPP5Bp. This protein is responsible for the majority of phosphatidyl (4,5) bisphosphate (PIP2) 5-phosphatase activity in platelets. Enzymatic assays demonstrate a deficiency of this same activity in Lowe patient\u27s fibroblast when compared to control fibroblasts. This activity suggests a role for the OCRL-1 protein (ocrl-1) in a signaling pathway, in particular, the inositol pathway. To characterize the OCRL-1 protein by immunological methods, antibodies were made to identify the protein. A single protein of 105 Kd was identified using affinity purified sera in western blot analysis of control fibroblast lysates and absent in the fibroblasts of a Lowe\u27s patient who lacked the OCRL-1 transcript. This antisera also recognizes a protein of similar mass in other human cell lines and in many mouse tissue homogenates. Immunofluorescence assays in fibroblast cells using the same purified sera, localize ocrl-1 to the Golgi complex. Brefeldin A (BFA) is a fungal product which reversibly disrupts the Golgi apparatus and recycles resident proteins back to the endoplasmic reticulum (ER). Studies demonstrate that ocrl-1 does not localize to the ER during BFA treatment but remains in a juxtanuclear position similar to proteins specifically localized to the trans-Golgi network (TGN) compartment of the Golgi complex. The recovery from BFA treatment comparing control and Lowe patient fibroblast cells demonstrates a marked difference in the time of Golgi reformation. These preliminary studies demonstrate an important difference which suggests that ocrl-1 may play an important role in vesicle formation. These immunological studies have identified and localized the protein defective in OCRL. Together with recent biochemical data, this information defines this protein as an inositol phospholipid enzyme localized to the Golgi complex with possible roles in vesicle formation. These studies can eventually aid in the elucidation of the precise role of ocrl-1, the pathways it affects and how the absence of ocrl-1 produces Lowe syndrome