Protein-tyrosine sulfation is mediated by one of only two Golgi tyrosylprotein sulfotransferases in the human and mouse genome (TPST-1 and TPST-2) that sulfate an unknown number of secreted and transmembrane proteins that transit the secretory pathway. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. However, only several dozen tyrosine-sulfated proteins have been described and for many, the functional importance of sulfotyrosine residues remains unknown. Many more tyrosine-sulfated proteins are likely to exist and await description. The pace of discovery of tyrosine-sulfated proteins has been very slow. There have been three major barriers to developing a full understanding of the importance of tyrosine sulfation in biological systems. First, there has been the lack of knockout animal models to study the biology of this enzyme system. Second, there has been the lack of a facile means to both identify additional tyrosine-sulfated proteins and probes to explore the role of tyrosine sulfation in protein function. Finally, a robust analytical method to determine the site of tyrosine sulfation in proteins has not been available. We developed Tpst1 and Tpst2 knockout mice and found that Tpst2-/- males are infertile, whereas Tpst1-/- males have normal fertility. The male infertility phenotype is characterized by normal spermatogenesis, but abnormal sperm function. These findings suggest that one or more proteins expressed in the male genital tract must be tyrosine-sulfated for normal sperm function. However, no proteins directly or indirectly involved in sperm function are known to be tyrosine- sulfated. We have developed a novel anti-sulfotyrosine monoclonal antibody that recognizes sulfotyrosine residues in proteins independent of the sequence context and which allows the detection and purification of tyrosine-sulfated proteins from complex biological samples. In addition, we have developed a novel subtractive mass spectrometry based method to determine the sites of tyrosine sulfation in proteins that is broadly applicable. We will exploit these novel tools and methods to identify tyrosine-sulfated proteins using the male genital tract as a model system in order to fully validate the approach, to refine our methodology, and to explore the role of tyrosine sulfation in male reproductive biology.
Three specific aims are proposed.
Aim 1 - Identify tyrosine-sulfated proteins expressed in the male genital tract. Expression profiles of tyrosine-sulfated proteins in the male genital tract of wild type, Tpst1-/-, and Tpst2-/- mice will be determined by PSG2 Western blotting in combination with subcellular fractionation. Tyrosine-sulfated proteins will then be purified from wild type mice by PSG2 affinity chromatography and identified by microsequencing.
Aim 2 The presence of sulfotyrosine and/or the site(s) of sulfation in the proteins identified in Aim 1 will be determined using our subtractive mass spectrometry strategy and other independent methodology.
Aim 3 Identify the protein(s) that are under sulfated in Tpst2-/- mice and the enzymatic basis for defective sulfation of these proteins.

Public Health Relevance

We propose to apply novel tools and methodology to identify tyrosine-sulfated proteins in the male genital tract. Our studies will catalyze studies in many areas of investigation by expanding the known TPST substrate repertoire, providing novel insights into structure-function relationships of proteins expressed in the male genital tract, and providing insights into the molecular basis for male infertility in Tpst2-/- mice. Our methods we will be broadly applicable to other system and/or model organism and facilitate a rapid expansion in our understanding of the role of tyrosine sulfation in biologic systems and human diseases.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD056022-05
Application #
8298668
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Moss, Stuart B
Project Start
2008-07-01
Project End
2013-09-30
Budget Start
2012-07-01
Budget End
2013-09-30
Support Year
5
Fiscal Year
2012
Total Cost
$299,566
Indirect Cost
$89,718
Name
Oklahoma Medical Research Foundation
Department
Type
DUNS #
077333797
City
Oklahoma City
State
OK
Country
United States
Zip Code
73104
Robinson, Michelle R; Moore, Kevin L; Brodbelt, Jennifer S (2014) Direct identification of tyrosine sulfation by using ultraviolet photodissociation mass spectrometry. J Am Soc Mass Spectrom 25:1461-71
Westmuckett, Andrew D; Nguyen, Edward B; Herlea-Pana, Oana M et al. (2014) Impaired sperm maturation in RNASE9 knockout mice. Biol Reprod 90:120
Nguyen, Edward B; Westmuckett, Andrew D; Moore, Kevin L (2014) SPACA7 is a novel male germ cell-specific protein localized to the sperm acrosome that is involved in fertilization in mice. Biol Reprod 90:16
Westmuckett, Andrew D; Siefert, Joseph C; Tesiram, Yasvir A et al. (2013) Salivary gland hypofunction in tyrosylprotein sulfotransferase-2 knockout mice is due to primary hypothyroidism. PLoS One 8:e71822
Kanan, Yogita; Hamilton, Robert A; Moore, Kevin L et al. (2012) Protein tyrosine-O-sulfation in bovine ocular tissues. Adv Exp Med Biol 723:835-41
Zarpellon, Alessandro; Celikel, Reha; Roberts, James R et al. (2011) Binding of alpha-thrombin to surface-anchored platelet glycoprotein Ib(alpha) sulfotyrosines through a two-site mechanism involving exosite I. Proc Natl Acad Sci U S A 108:8628-33
Marcello, Matthew R; Jia, Weitao; Leary, Julie A et al. (2011) Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm. J Biol Chem 286:13060-70
Westmuckett, Andrew D; Thacker, Kelly M; Moore, Kevin L (2011) Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo. PLoS One 6:e20406
Jen, Connie H; Leary, Julie A (2010) A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry. Anal Biochem 407:134-40
Danan, Lieza M; Yu, Zhihao; Ludden, Peter J et al. (2010) Catalytic mechanism of Golgi-resident human tyrosylprotein sulfotransferase-2: a mass spectrometry approach. J Am Soc Mass Spectrom 21:1633-42

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