Rapid assay to detect possible natural substrates of proteases in living cells.
Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.
Boonacker
Emil
E
University of Amsterdam, Amsterdam, The Netherlands.
Elferink
Sjoerd
S
Bardai
Abdennasser
A
Wormmeester
Jan
J
Van Noorden
Cornelis J
CJ
D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D004151:Dipeptides; D018819:Dipeptidyl Peptidase 4 / Q000032:analysis* / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D010078:Oxazines; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D004151:Dipeptides; D005456:Fluorescent Dyes; D010078:Oxazines; D012235:Rhodamines; C108786:alanyl-prolyl-cresyl violet; C475561:alanylproline-rhodamine 110; D018819:Dipeptidyl Peptidase 4