Sequence, Purification, and Cloning of an Intracellular Serine Protease, Quiescent Cell Proline Dipeptidase

Abstract

We recently observed that specific inhibitors of postproline 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.

Homodimerization via a Leucine Zipper Motif is Required for Enzymatic Activity of Quiescent cell Proline Dipeptidase

“…The post-proline cleaving enzymes are likely to emerge as an important protease family. As indicated above, CD26/DPPIV has been shown to modulate the function of several chemokines (3)(4)(5), while 1 Abbreviations: QPP, quiescent cell proline dipeptidase; DPPIV, dipeptidyl peptidase IV;…”

“…1 is a 58 kD protein that was recently isolated and cloned from human T cells (1). Highly specific inhibitors of post-proline cleaving aminodipeptidases cause cell death in quiescent lymphocytes, and the search for the target of these inhibitors led to the cloning of QPP and its subsequent nomenclature (2).…”

“…The post proline cleaving exopeptidases, QPP, PCP and CD26/DPPIV, have common structural features such as N-linked glycosylation and homodimerization that are important for their enzymatic activity (1,6,7,9,10,12). Leucine zipper motifs play an important role in the dimerization of a wide array of proteins (15)(16)(17)(19)(20)(21)(22).…”

“…The QPP constructs were cloned into the pCI-neo expression vector (Promega, WI), as previously described (1). The QPP-HA and QPP-myc constructs were generated by PCR with the high fidelity DeepVent polymerase (New England Biolabs, MA), using antisense primers incorporating either the myc (EQLLISEEDL) or the HA (YPYDVPDYA) epitope tags.…”

The intestinal contortin structure in Haemonchus contortus: An immobilised anticoagulant?

“…Catalysis by this type of peptidase is dependent on three active site residues, namely serine, histidine and aspartic acid which, although being dispersed throughout the primary protein sequence, come together in close proximity to form the active site when the enzyme is folded correctly. The catalytic residues in the Haemonchus PCP sequences here are ordered serine, aspartic acid and histidine and this feature, alongside the homology to PCPs place these enzymes in the clan SC, family S28 (Underwood et al, 1999). The region around the putative active site serine is highly conserved with 6/10 residues identical in all the comparators, this number diminishing to 3/10 and 1/8 around the putative active site aspartic acid and histidine, respectively.…”