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Post-translational processing of human procorticotrophin-releasing factor in transfected mouse neuroblastoma and
1School of Animal and Microbial Sciences, University of Reading, Berkshire, UK.
Abstract:
The mouse neuroblastoma cell line (Neuro 2 A) has been shown to contain the mRNA of a prohormone converting enzyme, PC2. The Chinese hamster ovary cell line (CHO) does not express PC2 mRNA, but is thought to contain the ubiquitous protease, furin. The enzyme(s) responsible for releasing corticotrophin-releasing hormone (CRH) from its precursor (proCRH) have not been identified, therefore to investigate the possible function(s) of PC2 or furin in the processing of proCRH, stable Neuro 2 A and CHO cell lines that express the 21 kDa human (h)proCRH were established. A specific two-site IRMA for CRH demonstrated that the hpreproCRH-expressing Neuro 2 A cell line cleaved the CRH precursor to the CRH peptide, and was able to release the mature peptide into cell medium at levels that were 4-fold higher than produced by the hproCRH-expressing CHO cells. RIA showed that the CHO cells secreted levels of CRH-containing peptides that were 10-fold higher than produced by the Neuro 2 A cells. Medium from the transfected CHO and Neuro 2 A cells was analysed by HPLC; this showed that CHO cells released a single protein corresponding to the unprocessed CRH precursor, whereas Neuro 2 A cells secreted two peptides, which could be identified as the 5 kDa CRH(1-41) and residual 16 kDa CRH peptides. These results suggest that Neuro 2 A cells, which contain PC2, can process proCRH to the mature peptide.
Insights
Mouse Neuro 2 A cells, expressing PC2, effectively process pro-corticotropin-releasing hormone (proCRH) into mature CRH. Chinese hamster ovary cells, expressing furin, showed less efficient proCRH processing, releasing mainly unprocessed precursor.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Cell Biology
Background:
- Prohormone convertase 2 (PC2) and furin are proteases involved in protein processing.
- The enzymes responsible for processing corticotropin-releasing hormone (CRH) precursor (proCRH) remain unidentified.
- Neuro 2 A cells express PC2, while CHO cells express furin.
Purpose of the Study:
- To investigate the role of PC2 and furin in proCRH processing.
- To compare the proCRH processing capabilities of Neuro 2 A and CHO cell lines.
Main Methods:
- Stable transfection of Neuro 2 A and CHO cells with human proCRH (hproCRH).
- Immuno-radiometric assay (IRMA) and radioimmunoassay (RIA) to quantify CRH peptide levels.
- High-performance liquid chromatography (HPLC) to analyze secreted peptides.
Main Results:
- Neuro 2 A cells processed hproCRH into mature CRH peptide and released it at higher levels than CHO cells.
- CHO cells secreted significantly higher levels of CRH-containing peptides, primarily unprocessed precursor.
- HPLC analysis confirmed Neuro 2 A cells secreted processed CRH (5 kDa and 16 kDa peptides), while CHO cells secreted unprocessed proCRH.
Conclusions:
- Neuro 2 A cells, containing PC2, demonstrate the ability to process proCRH into mature CRH.
- PC2 may play a significant role in the in vivo processing of proCRH.
- Furin in CHO cells appears less efficient in processing proCRH compared to PC2 in Neuro 2 A cells.