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M111901200v1
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Papers In Press, published online ahead of print March 18, 2002
J. Biol. Chem, 10.1074/jbc.M111901200
Submitted on December 13, 2001
Revised on March 18, 2002
Accepted on March 18, 2002

Clustered charged amino acids of human adenosine deaminase comprise a functional epitope for binding the adenosine deaminase complexing protein CD26/Dipeptidyl peptidase IV

Eva Richard, S. Munir Alam, Francisco X. Arredondo-Vega, Dhavalkumar D. Patel, and Michael S. Hershfield

Depts. of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710

Corresponding Author: msh{at}biochem.duke.edu

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 the function of CD26 as a costimulator of activated T cells. The CD26 binding site of human ADA has been localized by homolog scanning to the peripheral alpha 2 helix (amino acids 126-143). Among the 5 non-conserved residues within this segment, Arg142 in human and Gln142 in mouse ADA largely determined the capacity for stable binding to CD26 (Richard et al, J Exp Med 192:1223-35, 2000). We have now mutagenized conserved alpha 2 helix residues in human and mouse ADA, and used Surface Plasmon Resonance to evaluate binding kinetics to immobilized rabbit CD26. In addition to Arg142, we found that Glu139 and Asp143 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 KD for wild type human ADA from 17 nM 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.


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