Abstract
Human adenosine deaminase (ADA) occurs as a 41-kDa soluble monomer in all cells. On epithelia and lymphoid cells of humans, but not mice, ADA also occurs bound to the membrane glycoprotein CD26/dipeptidyl peptidase IV. This "ecto-ADA" has been postulated to regulate extracellular Ado levels, and also the function of CD26 as a co-stimulator of activated T cells. The CD26-binding site of human ADA has been localized by homolog scanning to the peripheral alpha2-helix (amino acids 126-143). Among the 5 non-conserved residues within this segment, Arg-142 in human and Gln-142 in mouse ADA largely determined the capacity for stable binding to CD26 (Richard, E., Arredondo-Vega, F. X., Santisteban, I., Kelly, S. J., Patel, D. D., and Hershfield, M. S. (2000) J. Exp. Med. 192, 1223-1235). We have now mutagenized conserved alpha2-helix residues in human and mouse ADA and used surface plasmon resonance to evaluate binding kinetics to immobilized rabbit CD26. In addition to Arg-142, we found that Glu-139 and Asp-143 of human ADA are also important for CD26 binding. Mutating these residues to alanine increased dissociation rates 6-11-fold and the apparent dissociation constant K(D) for wild type human ADA from 17 to 112-160 nm, changing binding free energy by 1.1-1.3 kcal/mol. This cluster of 3 charged residues appears to be a "functional epitope" that accounts for about half of the difference between human and mouse ADA in free energy of binding to CD26.
Highlights
Adenosine deaminase (ADA)1 catalyzes the hydrolytic deamination of adenosine (Ado) and 2Ј-deoxyadenosine
The latter has been identified as CD26/dipeptidyl peptidase IV (DPPIV), or CD26, a multifunctional membrane glycoprotein expressed on epithelia of liver, gut, kidney, and exocrine glands as well as on thymocytes and activated T lymphocytes [6, 7]
Studies involving alanine scanning mutagenesis have indicated that a subset of residues within interfaces makes a disproportionate contribution to binding energy (39 – 41)
Summary
Protein Reagents—CD26/DPPIV was purified from rabbit kidneys, and DPPIV activity was assayed, as described [23]. Analysis of ADA-CD26 Binding—Binding of [35S]Met-labeled ADA in vitro translation products to rabbit CD26/DPPIV (2 nmol/min DPPIV activity) during a 2-h incubation at 37 °C was analyzed by non-denaturing PAGE as reported previously [23]. Binding of recombinant ADA in lysates of E. coli SØ3834 to the CD26-expressing, ADA-deficient, HTLV-1-transformed AlNe human T lymphoblastoid cell line (LCL) was assessed by flow cytometry with the 1C5 mAb to human ADA as reported previously [23]. SPR analysis of ADA binding to purified rabbit kidney CD26/DPPIV was performed with a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden) For these studies, rabbit kidney CD26 and recombinant human CD4 glycoprotein (a nonspecific control) were coupled to the CMdextran surface of separate flow cells of the same Research Grade CM5 Sensor Chip, using amine coupling reagents provided by the manufacturer. Data are mean Ϯ S.D. from three or more experiments
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