A dipeptide metalloendoprotease substrate completely blocks the response of cells in culture to cholera toxin

Abstract

Prior exposure (15 min at 37 °C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mM N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH2, an inactive analogue of Cbz-Gly-Phe-NH2. The effect was reversible and dose- (IC50 as low as 0.2 mM depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH2 during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A1 peptide of CT and NAD+ activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH2-treated cells or when Cbz-Gly-Phe-NH2 was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH2 and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH2 on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca2+ homeostasis by Cbz-Gly-Phe-NH2 is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of 125I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH2 did not completely suppress the formation of the catalytically active A1 fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH2 cannot be excluded.