Application of peptides containing the cleavage sequence of pro-TNFalpha in assessing TACE activity of whole cells.
Tumor necrosis factor-alpha (TNFalpha) is presumably shed from cell membranes by TNFalpha-cleaving enzyme (TACE). The peptides SPLAQAVRSSSR and Dabcyl-LAQAVRSSSR-Edans, each encompassing the cleavage sequence of pro-TNFalpha recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus non-specific) of Dabcyl-TNFalpha-Edans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50 < 0.1 microM). HUVEC rapidly degraded TNFalpha-peptide, but in a non-specific manner (no SPLAQA detectable) and 50 microM TAPI-2 was without effect. Fluorescence was evoked when Dabcyl-LAQAVRSSSR-Edans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 microM TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of pro-TNFalpha cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive non-specific proteolytic cleavage by whole cells and cell extracts.
Becker
Bernhard F
BF
Department of Physiology, University of Munich, D-80336 Munich, Germany.
Gilles
Stefanie
S
Sommerhoff
Christian P
CP
Zahler
Stefan
S
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D005456:Fluorescent Dyes; D006321:Heart / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000201:enzymology; D008407:Mast Cells / Q000201:enzymology; D008666:Metalloendopeptidases / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D010455:Peptides; D011498:Protein Precursors; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha
D005456:Fluorescent Dyes; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C061153:tumor necrosis factor precursor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606464:Adam17 protein, rat