Dipeptidyl-peptidase IV-beta--further characterization and comparison to dipeptidyl-peptidase IV activity of CD26.
Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.
ERS 572 CNRS, Department SIDA/Retrovirus, Institut Pasteur, Paris, France.
J
J
Blanco
C
C
Nguyen
C
C
Callebaut
E
E
Jacotot
B
B
Krust
J P
JP
Mazaleyrat
M
M
Wakselman
A G
AG
Hovanessian
D019921:Biotinylation; D002460:Cell Line; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000737:chemistry; D010835:Phytohemagglutinins / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013205:Staphylococcal Protein A / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology
D004791:Enzyme Inhibitors; D037102:Lectins; D008562:Membrane Glycoproteins; D010835:Phytohemagglutinins; D013205:Staphylococcal Protein A; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D004492:Edetic Acid; D010450:Endopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100151:dipeptidyl peptidase II; D018819:Dipeptidyl Peptidase 4