Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro

Abstract

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5' and 3' untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers

“…Northern blotting confirmed a CPE transcript of approximately 2.4 kb, consistent with the predicted size of CPE-ΔN mRNA, different from the WT CPE transcript, which is 2.5 kb in size (ref. 29 and Figure 1C).…”

Enlargement of Secretory Vesicles by Protein Tyrosine Phosphatase PTP-MEG2 in Rat Basophilic Leukemia Mast Cells and Jurkat T Cells

“…1C). Double immunofluorescence staining with Abs against carboxypeptidase E, an enzyme typically located in secretory vesicles (30) where it processes prohormones by cleaving C-terminal arginine-containing motifs (31,32), showed a great deal of colocalization (Fig. 1, D and E), suggesting that PTP-MEG2 is primarily located on secretory vesicles.…”

A novel partner for the GTP‐bound forms of rho and rac

“…Overlay assay was performed according to Manser et al [14,18] using p21 loaded with [35S]GTP~,S or [35S]GDPflS (1000 Ci/mmol; New England Nuclear). After incubation with the probe (100 ng/ml), the blots were washed 5 times, 1 min each, in the described buffer [18].…”

“…After incubation with the probe (100 ng/ml), the blots were washed 5 times, 1 min each, in the described buffer [18].…”