Clustered Charged Amino Acids of Human Adenosine Deaminase Comprise a Functional Epitope for Binding the Adenosine Deaminase Complexing Protein CD26/Dipeptidyl Peptidase IV

Abstract

We have now mutagenized conserved ␣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 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.

Cell Surface Adenosine Deaminase Binds and Stimulates Plasminogen Activation on 1-LN Human Prostate Cancer Cells

“…1B) indicates a single class of binding sites (a K d of 18 Ϯ 5.4 nM and a B max of 0.8 nmol of bound ADA/nmol CD26). This K d value is within the same order of magnitude to previous estimates of K d determined for the binding of recombinant human ADA to purified rabbit CD26 (15).…”

“…It has been proposed that a lack of "ecto-ADA" contributes to severe combined immunodeficiency in patients with inherited ADA deficiency (10,11,13). Human ADA binds to CD26 via a carboxyl-terminal amino acid segment of the peripheral ␣2 helix, comprising residues 128 -143 (14,15). Binding of recombinant human ADA to CD26 was greatly reduced by mutating Arg142 to Gln, the amino acid occupying this position in mouse ADA, which does not bind CD26 (14).…”

Adenosine as an endogenous immunoregulator in cancer pathogenesis: where to go?

“…Similar findings regarding higher extracellular levels of adenosine in solid tumor microenvironment have also been confirmed by Ohta et al (2006) [63]. Besides being regulation of production of adenosine by CD39 and CD73 (described earlier), its levels are also controlled by dipeptidyl peptidase IV/CD26 [a binding protein for ADA] [64][65][66][67]. However, adenosine downregulates the expression of dipeptidyl peptidase IV/CD26 and its binding to ADA in colorectal carcinoma cells, which leads to further increase of extracellular adenosine levels in certain solid tumors [67].…”

Crystal Structure of CD26/Dipeptidyl-peptidase IV in Complex with Adenosine Deaminase Reveals a Highly Amphiphilic Interface

“…Although this lack of complex formation in rodents might be associated with amino acid substitutions in loops A and B of DPPIV and in helix ␣2 of ADA (Fig. 3), it has inspired mutational, binding, and kinetic studies on complex formation between DPPIV (18) and ADA (31).…”

“…The affinity of the complex between WT hADA and rDPPIV (K D ϭ 17 nM) is ϳ300ϫ stronger than between WT murine ADA (mADA) and rDPPIV (K D ϭ 5,400 nM). Compared with WT hADA, the most significant effects of single point mutations performed on helix ␣2 of hADA were found with Glu 139 3 Ala, Arg 142 3 Ala, and Asp 143 3 Ala, which showed an ϳ10-to 8-fold decrease in binding affinity to rDPPIV, with K D between 160 and 112 nM (31). In all cases, the kinetic constants for complex formation (k on ) were comparable (k on ϳ2 ϫ 10 4 M Ϫ1 s Ϫ1 ) and similar to that for WT hADA and rDPPIV, whereas those for complex dissociation differed by one power (k off ϳ2-4 ϫ 10 Ϫ3 s Ϫ1 ) compared with WT hADA, (k off ϳ4 ϫ 10 Ϫ4 s Ϫ1 .…”