Abstract
We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.
Highlights
There are relatively few enzymes that have the ability to cleave proline-containing peptide bonds
Northern Analysis—Total RNA was isolated from resting peripheral blood mononuclear cells (PBMCs) and Jurkat cells using the TRIZOL kit (Life Technologies). 20 g of total RNA per lane was loaded from each sample. 32P-labeled quiescent cell proline dipeptidase (QPP) cDNA was used to probe the Northern blot
Novel Intracellular DPPIV-like Activity in Lymphocytes— Functional analyses revealed that culturing PBMCs with CD26/DPPIV inhibitors led to apoptosis in resting lymphocytes [9]
Summary
Materials—The peptidase inhibitors Lys-thiazolidide, Lys-piperidide, and Val-boro-Pro (VbP) were provided by R. The soluble Ala-Pro-AFC cleaving activity of the QPP cDNA-transfected 293T human fibroblasts was partially purified by using a gel filtration column and an ion exchange column, in a similar manner as above. [3H]DFP Labeling—5 mg (total protein) of purified Ala-Pro-AFC cleaving activity was mixed with [3H]DFP (specific activity 8.4, Ci/ mmol) at a final concentration of 12 mM in 50 mM HEPES, pH 7.5, and incubated at room temperature for 60 min. The Ki for VbP was determined by assaying a standard amount of Ala-Pro-AFC cleaving activity and several concentrations of inhibitor. Transfection of QPP into 293T Fibroblasts—QPP cDNA was polymerase chain reaction-amplified with primers containing XhoI and EcoRI restriction sites using DeepVent polymerase (New England Biolabs) This was cloned into the pCI-neo expression vector (Promega) and transfected into 293T fibroblasts using the calcium phosphate method [11]. Northern Analysis—Total RNA was isolated from resting PBMCs and Jurkat cells using the TRIZOL kit (Life Technologies). 20 g of total RNA per lane was loaded from each sample. 32P-labeled QPP cDNA was used to probe the Northern blot
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