CPD-N is a
cytokine-inducible CPD (
carboxypeptidase-D)
isoform identified in rat Nb2 T-
lymphoma cells. The prototypic CPD (180 kDa) has three CP domains, whereas CPD-N (160 kDa) has an incomplete N-terminal domain I but intact domains II and III. CPD processes
polypeptides in the TGN (trans-Golgi network) but the Nb2 CPD-N is nuclear. The present study identified a cryptic exon 1', downstream of exon 1 of the rat CPD gene, as an alternative transcription start site that encodes the N-terminus of CPD-N. Western-blot analysis showed exclusive synthesis of the 160 kDa CPD-N in rat Nb2 and Nb2-Sp
lymphoma cells. Several haematopoietic cell lines including human K562 myeloma, Jurkat T-
lymphoma and murine CTLL-2 cytotoxic T-cells express a 160 kDa CPD-immunoreactive
protein, whereas mEL4 T-
lymphoma cells express the 180 kDa CPD. The CPD-immunoreactive
protein in hK562 cells is also nuclear and
cytokine-inducible. In contrast, MCF-7
breast cancer cells express only the 180 kDa CPD, which is mainly in the TGN. CPD/CPD-N assays using substrate
dansyl-L-alanyl-
L-arginine show approx. 98% of CPD-N activity in the Nb2 nucleus, whereas MCF-7 CPD activity is enriched in the post-nuclear 10000 g pellet. The K(m) for CPD-N and CPD are 132+/-30 and 63+/-9 microM respectively. Specific activity/K(m) ratios show that
dansyl-L-alanyl-
L-arginine is a better substrate for CPD-N than for CPD. CPD-N has an optimal pH of 5.6 (due to domain II), whereas CPD has activity peaks at pH 5.6 (domain II) and pH 6.5-7.0 (domain I). CPD and CPD-N are inhibited non-competitively by
zinc chelator 1,10-phenanthroline and competitively by
peptidomimetic inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic
acid. The Nb2 CPD-N co-immunoprecipitated with
phosphatase PP2A (
protein phosphatase 2A) and alpha4
phosphoprotein. In summary, a
cytokine-inducible CPD-N is selectively expressed in several haematopoietic tumour cells. Nuclear CPD-N is enzymatically active and interacts with known partners of CPD.