A novel 40kDa N-terminal truncated carboxypeptidase E splice variant: cloning, cDNA sequence analysis and role in regulation of metastatic genes in human cancers.

Xuyu Yang,Y Peng Loh,Hong Lou,Shui-Feng Huang,Ya-Ting Chen

doi:10.18632/genesandcancer.193

Abstract

Carboxypeptidase E (CPE), a prohormone processing enzyme, is a 476- amino acid protein with a signal peptide in its N-terminus and is expressed in the nervous and the endocrine systems. Recent evidence indicate CPE plays various non-enzymatic roles in the endocrine and nervous systems and in various cancers. Besides wild type (WT) CPE, a 40-kDa CPE protein that localizes in the nucleus and cytoplasm has been described in embryonic mouse brain. In this study we have cloned this CPE variant encoding the 40kDa CPE-ΔN protein from human cancer cells. RACE assay and sequence analysis confirmed existence of this CPE variant mRNA, which has 198 nucleotides removed within the first exon and 589 nucleotides from the 3’-UTR, respectively, compared to WT-CPE mRNA. Bioinformatic analysis revealed that this CPE variant mRNA has a shortened open reading frame, which starts coding from the 3rd ATG relative to WT-CPE mRNA and encodes a 40kDa N-terminus truncated CPE protein. RT-PCR and Western blot analysis showed that 40kDa CPE-ΔN is expressed in multiple cancer cell lines and tumor tissues. Overexpression of this 40kDa CPE-ΔN variant up-regulated expression of multiple metastatic genes encompassing different signaling pathways, suggesting potentially an important role of CPE-ΔN in tumor metastasis.

Highlights

Carboxypeptidase E (CPE), known as carboxypeptidase H (CPH), is a prohormone processing enzyme that is encoded by the human CPE gene [1]
We observed that the liver cancer line, HCC97H, required a large amount of mRNA input compared with the other cancer cell lines for detection of both CPE transcripts, indicating a relative lower abundance of CPE transcripts within those cells
Of the two transcripts detected in all the various cancer lines studied, the upper one in the Northern blot appears to be ~2.4kb in size, which is approximately the same size as described by Lim et al [1] and we identified this one as the wild type (WT)-CPE transcript; the lower band, has never been described previously

Summary

Introduction

Carboxypeptidase E (CPE), known as carboxypeptidase H (CPH), is a prohormone processing enzyme that is encoded by the human CPE gene [1]. CPE is a 476- amino acid protein with a signal peptide in its N-terminus that is mainly expressed in brain and throughout the neuroendocrine system, including the endocrine pancreas, pituitary, and adrenal gland chromaffin cells [2]. CPE contains several functional domains, a signal peptide directing the protein into the rough endoplasmic reticulum (RER) cisternae at the N-terminus, a catalytic domain in the middle, and a highly acidic C-terminal domain [7, 8]. Aberrant expression of CPE has been found in several major tumors of epithelial origin, including lung, liver, colon, pancreatic and cervical cancers [13, 14, 15, 16, 17], as well as in neuroendocrine tumors such as insulinoma [3, 18], suggesting CPE might have a role in tumor progression. In glioblastoma (GBM) cells, CPE has been identified as a regulator of RPS6 within the mTORc1 signaling pathway to reduce aerobic glycolysis and migration, which negatively affects tumor cell invasion and migration [20]; in contrast, CPE may promote migration and invasion in other tumor types such as cervical cancer and osteosarcoma [17, 21], the underlying mechanism remains to be elucidated

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Results

Conclusion