Abstract
Dipeptidyl peptidase I (DPPI) is a cysteine protease found predominantly in myelomonocytic cells, cytotoxic T-cells, and mast cells. Recent studies identify an intracellular role for mast cell-DPPI (MC-DPPI) by activating prochymase and protryptase to their mature forms. To better define MC-DPPI and to explore the possibility of extracellular roles, we purified MC-DPPI from mastocytoma cells. We found the dog C2 mastocytoma cell line to be the richest source yet described for DPPI, purifying up to 200 microg of enzyme per g of cells. Dog MC-DPPI has an Mr of approximately 175,000 and consists of four subunits, each composed of a propeptide, light chain, and heavy chain. The heavy chain is N-glycosylated and is heterogeneously processed to three different forms. NH2-terminal sequences of the heavy chain and propeptide are identical to those predicted from a cDNA clone we sequenced from a mastocytoma cDNA library. The dog cDNA-derived sequence is 86% identical to that of human DPPI. Dog mastocytoma cells incubated with 12-O-tetradecanoylphorbol-13-acetate increase expression of MC-DPPI mRNA. MC-DPPI maintains its activity for dipeptide substrates at a neutral to alkaline pH. Cells stimulated with ionophore or substance P secrete MC-DPPI in parallel with the granule-associated mediators tryptase and histamine. Thus, dog mastocytoma cells secrete DPPI that is active at the pH of extracellular fluids, suggesting that MC-DPPI may act outside the cell.
Highlights
The C1 family of cysteine proteases includes the lysosomal proteases cathepsins B, C, H, S, and L
Human Dipeptidyl peptidase I (DPPI) is processed into a mature, proteolytically active enzyme consisting of the Mr 23,000 heavy chain, the Mr 7000 light chain, and an Mr 16,000 propeptide that remains associated with the active enzyme
Dog mast cell-DPPI (MC-DPPI) is similar to human DPPI in oligomeric structure, activity at acidic pH, inhibition by inactivators of cysteine proteinases, and inducibility of mRNA expression [15, 27, 32]
Summary
Materials—All chemicals are from Sigma unless otherwise specified. Cell Culture—Dog BR and C2 mastocytoma cells were a gift of S. Purification of DPPI—C2 cell pellets were resuspended in 100 mM sodium acetate, 300 mM NaCl, 1 mM EDTA (pH 6.0) (Buffer A), sonicated, and centrifuged at 10,000 ϫ g for 15 min. 15 l of enzyme solution were incubated in 0.2 ml of 0.06 M Tris-HCl (pH 7.8) containing 0.4% dimethyl sulfoxide and 80 g/ml of substrate at 37 °C In both enzyme assays, release of free nitroaniline was measured at 410 nm for 5–10 min. Mastocytoma cells were harvested by centrifugation, washed twice in Ca2ϩ- and Mg2ϩ-free phosphate-buffered saline, and resuspended in serum-free Dulbecco’s modified Eagle’s-H16 medium to a final concentration of 15 ϫ 106 cells/ml. Activity was measured using L-Ala-Ala-p-nitroanilide as substrate for MC-DPPI
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