Post-translational processing of human procorticotrophin-releasing factor in transfected mouse neuroblastoma and Chinese hamster ovary cell lines.
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.
Brar
B
B
School of Animal and Microbial Sciences, University of Reading, Berkshire, UK.
Sanderson
T
T
Wang
N
N
Lowry
P J
PJ
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015592:Immunoradiometric Assay; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000378:metabolism*; D011863:Radioimmunoassay; D013381:Subtilisins / Q000378:metabolism*
D011498:Protein Precursors; C041645:pro-corticotropin releasing hormone; D003346:Corticotropin-Releasing Hormone; D013381:Subtilisins; D045706:Proprotein Convertase 2