Clustered charged amino acids of human adenosine deaminase comprise a functional epitope for binding the adenosine deaminase complexing protein CD26/dipeptidyl peptidase IV.
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.
Richard
Eva
E
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Alam
S Munir
SM
Arredondo-Vega
Francisco X
FX
Patel
Dhavalkumar D
DD
Hershfield
Michael S
MS
D000243:Adenosine Deaminase / Q000737:chemistry*; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D015153:Blotting, Western; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors
D018076:DNA, Complementary; D000939:Epitopes; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D018819:Dipeptidyl Peptidase 4; D000243:Adenosine Deaminase