Retinal degeneration caused by alterations in protein O-sulfation

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The overall goal of this proposal is to investigate the role of protein tyrosyl-sulfation in normal retinal function. The intermediate aims include the identification of retinal proteins that are sulfated by tyrosylprotein sulfotransferases and determine how lack of either (or both) of these enzymes influence retinal function. There are two distinct TPST isoenzymes present in most tissues. These two enzymes are responsible for the post-translational tyrosine O-sulfation of cellular proteins. Neither these enzymes nor their targets have been studied in the retina or other ocular tissues. We have generated knockout mice for TPST-1 & 2. These two mice, and the double knockout obtained by crossbreeding, exhibit different retinal defects in retinal development, function and aging. Lack of TPST-1 results in the retardation of functional development. Moreover, these mice demonstrate a slow retinal degeneration characterized by a gradual loss of scotopic and photopic electroretinography (ERGs) that become very apparent at 4 months of age (-30% loss). Histologic examination of P120 Tpstrl- retinas revealed a reduced number of nuclei in ONL (9 rows at P120 versus 11 rows at P50) and INL (4 rows at P120 versus 5-6 at P50). Unlike TpsWI - retinas, TpstZI - retinas never fully develop to the wild-type level. This very clearly demonstrates a direct role for TPST-2, or its protein products, in retinal maturity. In Aim 1, we will characterize the presence, localization and biochemical behavior of retinal TPST-1 and TPST-2 during development. These experiments will be performed in normal mouse retinas from embryonic day 10 to 2 years of age utilizing antibodies that are specific for TPST-1, TPST-2, and to sulfotyrosine. As part of this aim, we will identify the proteins modified by TPST-1/2 using classic immunoaffinity purification techniques and MS and MS/MS. In Aim 2, we will fully characterize retinal phenotypes caused by lack of TPST-1, TPST-2 or both. Characterization will be performed structurally at the light and electron microscopic levels, functionally by scotopic and photopic ERGs and suction electrode recordings, and biochemically by analysis of the sulfation of retinal proteins and expression of the targets of TPST-1/2 by SOS-PAGE. These studies will, for the first time, shed light on the role of TPST-1 and 2 in protein sulfation in the retina. Further, they will increase our understanding of retinal development and the mechanism(s) involved in retinal degeneration.