Transcellular Proteolysis Demonstrated by Novel Cell Surface-associated Substrates of Dipeptidyl Peptidase IV (CD26)

Abstract

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 surfacebound 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.

Activatable Optical Probes for the Detection of Enzymes

“…screened a range of haloalkane and maleimide groups as electrophilic handles for proteins, with their optimal design permitting the assessment of dipeptidyl peptidase activity in single cells [287]. A limitation of these probes is their reduced sensitivity due to the incomplete recovery of fluorescence from the cleavage of just one of the N-acyl groups.…”

Enzymatic activity of the SARS coronavirus main proteinase dimer

“…Upon cleavage by an endoproteinase of one of the two amide bonds adjacent to the Rhodamine moiety, the Rhodamine moiety in the resultant mono‐substituted product switches to the quinone state exhibiting a high degree of conjugation; concomitant with this is a massive increase in fluorescence intensity. Rhodamine‐based substrates have been used to assay caspases in vitro [12] and in vivo; trypsin, plasmin and thrombin [8]; leucine aminopeptidase and dipeptidyl aminopeptidase in solution [13] and on the cell surface [14]; cathepsin K in vivo expressed in CHO cells [15]; elastase [16]; and the adenovirus proteinase [10,11].…”

Fluorogenic Substrate [Ala-Pro]2-Cresyl Violet But Not Ala-Pro-Rhodamine 110 Is Cleaved Specifically by DPPIV Activity: A Study in Living Jurkat Cells and CD26/DPPIV-transfected Jurkat Cells

“…Permeabilization of cells almost doubled the production of fluorescence, indicating that diffusion into the cells of the rhodamine 110-based substrate is a limiting step in the accessibility of this substrate for intracellular proteases other than CD26/DPPIV. Therefore, we conclude that the nature of the fluorophore significantly affects interactions of these synthetic substrates with the active site of DPPIV as has been demonstrated before (Chase and Shaw 1969;Wong and Shaw 1976;Castillo et al 1979;Lorey et al 2002). …”

“…Rhodamine 110 is not fluorescent when amino acids are attached, as it is completely quenched (Leytus et al 1983a) and becomes fluorescent after proteolytic release, whereas cresyl violet is always fluorescent even when amino acid sequences are attached but fluorescence is shifted to a longer wavelength when the amino acid sequences are removed (Boonacker and Van Noorden 2001). However, the amino acid sequence does not completely determine substrate specificity because intrinsic chemical properties of fluorophores in synthetic substrates may also affect the reactivity of the substrate with the protease (Chase and Shaw 1969;Boonacker and Van Noorden 2001;Lorey et al 2002). This phenomenon can be due to steric hindrance or to particular chemical properties of the fluorophore.…”