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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY018137-02
Application #
7843629
Study Section
Special Emphasis Panel (ZRG1-CB-G (90))
Program Officer
Neuhold, Lisa
Project Start
2009-06-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
2
Fiscal Year
2010
Total Cost
$371,400
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
878648294
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I (2016) The Potential Role of Flavins and Retbindin in Retinal Function and Homeostasis. Adv Exp Med Biol 854:643-8
Kanan, Yogita; Al-Ubaidi, Muayyad R (2016) Identification of Tyrosine O Sulfated Proteins in Cow Retina and the 661W Cell Line. Adv Exp Med Biol 854:649-54
Kanan, Yogita; Al Ubaidi, Muayyad R (2015) Detection of tyrosine sulfation on proteins. Curr Protoc Protein Sci 80:14.7.1-20
Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I (2015) Retbindin is an extracellular riboflavin-binding protein found at the photoreceptor/retinal pigment epithelium interface. J Biol Chem 290:5041-52
Han, Zongchao; Banworth, Marcellus J; Makkia, Rasha et al. (2015) Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype. FASEB J 29:2535-44
Kanan, Y; Al-Ubaidi, M R (2015) Role of tyrosine-sulfated proteins in retinal structure and function. Exp Eye Res 133:126-31
Murray, Anne R; Vuong, Linda; Brobst, Daniel et al. (2015) Glycosylation of rhodopsin is necessary for its stability and incorporation into photoreceptor outer segment discs. Hum Mol Genet 24:2709-23
Kanan, Y; Gordon, W C; Mukherjee, P K et al. (2015) Neuroprotectin D1 is synthesized in the cone photoreceptor cell line 661W and elicits protection against light-induced stress. Cell Mol Neurobiol 35:197-204
Al-Ubaidi, Muayyad R (2014) RGC-5: are they really 661W? The saga continues. Exp Eye Res 119:115
Kanan, Yogita; Brobst, Daniel; Han, Zongchao et al. (2014) Fibulin 2, a tyrosine O-sulfated protein, is up-regulated following retinal detachment. J Biol Chem 289:13419-33

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