Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.
Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.
Marriott
D
D
Department of Cell Genetics, Genentech, Inc, South San Francisco, California 94080.
Gillece-Castro
B
B
Gorman
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005656:Fungal Proteins / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics / Q000494:pharmacology; D010911:Pituitary Neoplasms / Q000201:enzymology; D045664:Proprotein Convertase 1; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011994:Recombinant Proteins / Q000494:pharmacology; D012065:Relaxin / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000494:pharmacology*; D013381:Subtilisins
D005656:Fungal Proteins; D009363:Neoplasm Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C038656:prorelaxin; D012065:Relaxin; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1