Pro-thyrotropin-releasing hormone processing by recombinant PC1.
Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [3H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH25-152 or pre-proTRH25-158) and a 10-kDa C-terminal peptide (pre-proTRH154-255 or preproTRH160-255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (prepro-TRH25-76 or preproTRH25-82) and a 3.8-kDa peptide (preproTRH83-108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (prepro-TRH206-255). The optimal pH for these cleavages was 5.5. ZnCl2, EDTA, EGTA, and the omission of Ca2+ inhibited the formation of pYE27 (preproTRH25-50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.
Nillni
E A
EA
Department of Medicine, Brown University, Rhode Island Hospital, Providence 02903, USA.
Friedman
T C
TC
Todd
R B
RB
Birch
N P
NP
Loh
Y P
YP
Jackson
I M
IM
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D011994:Recombinant Proteins; D013973:Thyrotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism*; D013997:Time Factors; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D011480:Protease Inhibitors; D011498:Protein Precursors; D011994:Recombinant Proteins; D017967:Zinc Compounds; D013973:Thyrotropin-Releasing Hormone; C040476:thyrotropin-releasing hormone, Gly; C016837:zinc chloride; C046418:pro-thyrotropin releasing hormone; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases; D011761:Pyrrolidonecarboxylic Acid