Biochemical effects of adenosine (Ado) and 2'-deoxyadenosine (dAdo) block lymphoid development and cause severe immune dysfunction in genetic deficiency of Ado deaminase (ADA). One target of these nucleosides is the essential metabolic enzyme S-adenosylhomocysteine hydrolase (AdoHcyase), which we identified as a high affinity Ado and cyclic AMP binding protein. Ado inhibits AdoHcy hydrolysis in ADA deficient cells. We discovered that dAdo irreversibly inactivates AdoHcyase by causing reduction of enzyme associated NAD. In ADA deficiency these effects of Ado and dAdo result in secondary deficency of AdoHcyase and accumulation of AdoHcy, which is an inhibitor of S-adenosylmethionine (AdoMe+) dependent transmethylation reactions. We recently showed that the genetic loci for AdoHcyase and ADA are linked, suggesting an evolutionary relationship between these enzymes. We propose to examine the possible relatedness of AdoHycase to ADA or to other proteins that bind NAD, Ado and cAMP; to provide information, which is currenty unavailable, regarding the primary sequence and structure of AdoHcyase and the nature of its catalytic and ligand binding sites; to investigate the influence of ADA deficiency on the turnover of AdoHcyase; and to continue studies now in progess of the metabolic consequences to lymphoid cells of the inhibition of AdoHcy hydrolysis. Specifically, we will attempt to isolate and sequence the cDNA for human AdoHcyase mRNA, using monoclonal antibodies to AdoHcyase and AdoHcyase specific oligonucleotides as probes for screening human liver cDNA libraries. We will evaluate the possibility that Adohcyase may be composed of two kinds of subunits. Essential amino acid residues involved in ligand binding and in catalysis will be located within the sequence of AdoHcyase. We will attempt to crystallize AdoHcyase in the presence and absence of ligands so that its structure may be established definitively. Antibodies and cDNA probes will be used to determine whether inactivation of AdoHcyase by dAdo in ADA deficiency alters the rate of AdoHCyase degradation or AdoHcyase turnover. We will characterize lymphoblast mutants that we have selected for resistance to Ado analogues that specifically inhibit Adohcyase, with the aim of better defining the effect of AdoHcy accumulation on the synthesis and turnover of AdoMet.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK020902-09A1
Application #
3226832
Study Section
Biochemistry Study Section (BIO)
Project Start
1978-01-01
Project End
1991-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
9
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Liu, Ping; Santisteban, Ines; Burroughs, Lauri M et al. (2009) Immunologic reconstitution during PEG-ADA therapy in an unusual mosaic ADA deficient patient. Clin Immunol 130:162-74
Alangari, Abdullah; Al-Harbi, Abdullah; Al-Ghonaium, Abdulaziz et al. (2009) Purine nucleoside phosphorylase deficiency in two unrelated Saudi patients. Ann Saudi Med 29:309-12
Gracia, Eduard; Cortes, Antoni; Meana, J Javier et al. (2008) Human adenosine deaminase as an allosteric modulator of human A(1) adenosine receptor: abolishment of negative cooperativity for [H](R)-pia binding to the caudate nucleus. J Neurochem 107:161-70
Engel, Barbara C; Podsakoff, Greg M; Ireland, Joanna L et al. (2007) Prolonged pancytopenia in a gene therapy patient with ADA-deficient SCID and trisomy 8 mosaicism: a case report. Blood 109:503-6
Malacarne, Fabio; Benicchi, Tiziana; Notarangelo, Lucia Dora et al. (2005) Reduced thymic output, increased spontaneous apoptosis and oligoclonal B cells in polyethylene glycol-adenosine deaminase-treated patients. Eur J Immunol 35:3376-86
Lainka, Elke; Hershfield, Michael S; Santisteban, Ines et al. (2005) polyethylene glycol-conjugated adenosine deaminase (ADA) therapy provides temporary immune reconstitution to a child with delayed-onset ADA deficiency. Clin Diagn Lab Immunol 12:861-6
Chan, Belinda; Wara, Diane; Bastian, John et al. (2005) Long-term efficacy of enzyme replacement therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). Clin Immunol 117:133-43
Blanchet, Fabien; Cardona, Ana; Letimier, Fabrice A et al. (2005) CD28 costimulatory signal induces protein arginine methylation in T cells. J Exp Med 202:371-7
Gonzalez-Gronow, Mario; Hershfield, Michael S; Arredondo-Vega, Francisco X et al. (2004) Cell surface adenosine deaminase binds and stimulates plasminogen activation on 1-LN human prostate cancer cells. J Biol Chem 279:20993-8
Myers, Laurie A; Hershfield, Michael S; Neale, Wirt T et al. (2004) Purine nucleoside phosphorylase deficiency (PNP-def) presenting with lymphopenia and developmental delay: successful correction with umbilical cord blood transplantation. J Pediatr 145:710-2

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