Inhibition of dipeptidyl peptidase I in the human mast cell line HMC-1: blocked activation of tryptase, but not of the predominant chymotryptic activity.
The mast cell proteases tryptase and chymase are synthesised as inactive precursors, but are stored and secreted as active enzymes. The cysteinyl protease dipeptidyl peptidase I (DPPI, cathepsin C) can activate the corresponding proenzymes in cell-free systems, but it is unknown whether it fulfils this role within the intact cell. We, therefore, tested the effect the DPPI-selective inhibitor Gly-Phe diazomethyl ketone (Gly-Phe-CHN(2)) on the tryptic and chymotryptic activity of the human mast cell-like cell line, HMC-1, and monitored any changes in the amount of immunodetectable enzymes by flow cytometry. Culture in Gly-Phe-CHN(2) produced a significant decrease in tryptase activity in cell lysates within 24hr and further decreases during continued culturing to 216 hr with periodic replenishment of Gly-Phe-CHN(2)-containing media. Flow cytometry showed no significant change in the levels of immunoreactive tryptase. In contrast, chymotryptic activity in treated cells did not differ significantly from untreated cells at any time point. Treatment of 216 hr cell lysates with DPPI revealed significant amounts of activatable protryptase in Gly-Phe-CHN(2)-treated cells, but not in controls, whereas activatable prochymotryptic activity was found in both treated and control cells. Chymase was detected immunologically, though small differences in substrate specificity and molecular mass were observed. These results strongly suggest that DPPI plays a role in the activation of tryptase, but not of the predominant chymotryptic activity of HMC-1 cells. As inhibitors of tryptase have proven efficacious in models of allergic disease, these results also indicate that inhibitors of DPPI might provide an additional point of therapeutic control.
Sheth
Parimal D
PD
Immunopharmacology Group, Division of Infection, Immunity and Repair, University of Southampton Medical School, Southampton General Hospital, Southampton SO16 6YD, UK.
Pedersen
John
J
Walls
Andrew F
AF
McEuen
Alan R
AR
D000818:Animals; D020789:Cathepsin C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism*; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008407:Mast Cells / Q000187:drug effects* / Q000201:enzymology*; D011817:Rabbits; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D053802:Tryptases
D004151:Dipeptides; D015842:Serine Proteinase Inhibitors; C089960:glycylphenylalanine diazomethyl ketone; D003978:Diazomethane; D020789:Cathepsin C; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D053802:Tryptases