The primary focus of the present proposal is on how the machinery for sulfation, a common posttranslational modification of proteins, lipids and carbohydrates (especially proteoglycans) is organized and controlled in higher organisms. The integrated pathway for sulfate uptake, activation and utilization encompasses multiple components and multiple intracellular compartments. Central to this process is the bifunctional PAPS synthetase which synthesizes phosphoadenosyiphosphosulfate (PAPS) from ATP and sulfate- in a two-step reaction. Recent work, including the discovery of the PAPS synthetase gene family, the identification of mutations in PAPS synthetase that lead to both human and animal chondrodystrophies and elucidation of unique enzymatic properties, underlines the significance of this enzyme in the overall sulfation process. Two specific questions will be addressed in this continuation proposal. 1) What features of the fused bifunctional PAPS synthetase and individual monofunctional sulfurylases and kinases account for the significant mechanistic differences between them, especially with respect to the channeling phenomenon? Mutagenic analysis and structural elucidation approaches will be employed. 2) What is the role of the multiple PAPS synthetase family members with respect to tissue- and developmental-specific expression patterns? A multi-faceted approach will be used to quantitate each isoform, elucidate mechanisms of transcriptional regulation and determine the consequences of manipulation of isoform expression. These studies are aided by the availability of a mutant model system with a defect in sulfation that results in altered proteoglycan production and abnormal skeletal growth and development. The range of methodology necessary for accomplishing these goals includes: cDNA cloning and sequencing, enzyme assay and kinetic analysis, cell and tissue culture and propagation of the mutant mouse strain. These studies have the long-term goal of providing a model of how this critically important pathway is regulated and then to correlate defects in the overall pathway with abnormal growth and development.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Pathobiochemistry Study Section (PBC)
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Vitkovic, Ljubisa
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University of Chicago
Schools of Medicine
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Cortes, Mauricio; Cortes, Leslie K; Schwartz, Nancy B (2015) Mapping proteoglycan functions with glycosidases. Methods Mol Biol 1229:443-55
Lauing, Kristen L; Cortes, Mauricio; Domowicz, Miriam S et al. (2014) Aggrecan is required for growth plate cytoarchitecture and differentiation. Dev Biol 396:224-36
Schwartz, Nancy B; Domowicz, Miriam S (2014) Chemistry and function of glycosaminoglycans in the nervous system. Adv Neurobiol 9:89-115
Mis, Emily K; Liem Jr, Karel F; Kong, Yong et al. (2014) Forward genetics defines Xylt1 as a key, conserved regulator of early chondrocyte maturation and skeletal length. Dev Biol 385:67-82
Bradley, Michael E; Rest, Joshua S; Li, Wen-Hsiung et al. (2009) Sulfate activation enzymes: phylogeny and association with pyrophosphatase. J Mol Evol 68:1-13
Domowicz, Miriam S; Cortes, Mauricio; Henry, Judith G et al. (2009) Aggrecan modulation of growth plate morphogenesis. Dev Biol 329:242-57
Pirok 3rd, Edward W; Domowicz, Miriam S; Henry, Judith et al. (2005) APBP-1, a DNA/RNA-binding protein, interacts with the chick aggrecan regulatory region. J Biol Chem 280:35606-16
Singh, Bhawani; Schwartz, Nancy B (2003) Identification and functional characterization of the novel BM-motif in the murine phosphoadenosine phosphosulfate (PAPS) synthetase. J Biol Chem 278:71-5
Kurima, K; Singh, B; Schwartz, N B (1999) Genomic organization of the mouse and human genes encoding the ATP sulfurylase/adenosine 5'-phosphosulfate kinase isoform SK2. J Biol Chem 274:33306-12
Deyrup, A T; Krishnan, S; Singh, B et al. (1999) Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase. J Biol Chem 274:10751-7

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