Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26).
Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.
Lorey
Susan
S
Department of Biochemistry and Biotechnology, Institute of Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle (Saale), Germany. lorey@biochemtech.uni-halle.de
Faust
Jurgen
J
Mrestani-Klaus
Carmen
C
Kähne
Thilo
T
Ansorge
Siegfried
S
Neubert
Klaus
K
Bühling
Frank
F
D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D002450:Cell Communication; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D011485:Protein Binding; D012235:Rhodamines / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection; D020298:U937 Cells
D005456:Fluorescent Dyes; D012235:Rhodamines; C049025:rhodamine 110; D018819:Dipeptidyl Peptidase 4