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Originally published In Press as doi:10.1074/jbc.M111901200 on March 18, 2002

J. Biol. Chem., Vol. 277, Issue 22, 19720-19726, May 31, 2002
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Clustered Charged Amino Acids of Human Adenosine Deaminase Comprise a Functional Epitope for Binding the Adenosine Deaminase Complexing Protein CD26/Dipeptidyl Peptidase IV*

Eva RichardDagger §, S. Munir AlamDagger , Francisco X. Arredondo-VegaDagger , Dhavalkumar D. PatelDagger , and Michael S. HershfieldDagger ||**

From the Departments of Dagger  Medicine,  Immunology, and || Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

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 alpha 2-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 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 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 KD 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.

* This work was supported in part by Grants RO1 DK20902 (to M. S. H.) and R01 AI47604 (to D. D. P.) from the National Institutes of Health, by a grant from Enzon, Inc. (to M. S. H.), and by a Scientist Development grant from the American Heart Association (to S. M. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Fellowship 98/9329 from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain.

** To whom correspondence and reprint requests should be addressed: Box 3049, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-4184; Fax: 919-684-4168; E-mail: msh@biochem.duke.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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