Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease.

Abstract

During male gonadal development Mullerian duct regression is mediated by the actions of the hormone Mullerian inhibiting substance (MIS), a member of the transforming growth factor 13 superfamily. MIS is considered to be unique among members of this superfamily because bioactivation of MIS via proteolytic processing is hypothesized to occur at its target organ, the Mullerian duct. We find instead that the majority of MIS is processed and secreted from the embryonic testes as a complex in which the mature region remains noncovalently associated with the the prodomain. In addition, we have identified two candidate endoproteases that are expressed in the testes and that may be capable of processing MIS in vivo. These kex2/subtilisin-like enzymes, PCS and furin, are members of the proprotein convertase family that have been implicated in hormone bioactivation via proteolytic processing after dibasic amino acid cleavage recognition sites. Coexpression of PC5 and MIS in transfected mammalian cells results in efficient processing and bioactivation of MIS. Our results suggest that MIS is a natural substrate for PC5, thereby supporting a role for prohormone convertases in the activation of transforming growth factor 13-related hormones during development.Mammalian male sexual differentiation requires maintenance of the male reproductive tract with simultaneous regression of the female reproductive tract, specifically the Mullerian duct (1). A key regulator of this morphological regression is Mullerian inhibiting substance (MIS), which is expressed and secreted as a homodimer by the Sertoli cells of the developing rat testis commencing at embryonic day (E) 13. It has been assumed that MIS bioactivation requires proteolytic cleavage of the prohormone at a monobasic site (Arg-Ala-Gln-Arg) into its NH2-terminal proregion and bioactive COOH-terminal fragment. Indeed, Cate and coworkers demonstrated bioactivity of MIS after in vitro cleavage at this site with the serine protease plasmin (2). However, unlike ,B transforming growth factor 3 (TGF-P), where secretion and cleavage are tightly linked, it has been proposed that the majority of MIS is cleaved after reaching its target site (Mullerian duct; refs. 3 and 4). Thus, the enzymes responsible for cleavage in vivo are thought to reside within the mesonephros containing the Mullerian duct. Here we examine the nature and mechanism of MIS bioactivation during reproductive tract development.In searching for candidate processing enzymes of MIS, our attention focused on members of the proprotein convertase gene family shown to process large precursor proteins at both di-and monobasic cleavage sites. Initial identification of the first hormone processing enzyme came from genetic analyses of yeast kex2 mutants that are sterile because they are unable to process the yeast mating pheromone a-factor (5). Later it was shown that the kex2 gene product encodes for a subtilisinlike serine protease. The search for mammalian homologues has led to the identification of six kex2/subtilis...

In Vitro Cleavage of Internally Quenched Fluorogenic Human Proparathyroid Hormone and Proparathyroid-related Peptide Substrates by Furin

“…The list of such proteins includes the pro-Mü llerian inhibiting substance, proneurokinin A, propancreatic polypeptide, and proprotein tyrosine phosphatase-receptor. Both pro-Mü llerian inhibiting substance and proprotein tyrosine phosphatase-receptor are cleaved by PC5 (41,42), and propancreatic polypeptide is processed in the regulated secretory pathway by the neuroendocrine-specific enzymes, PC1 and PC2 (43). As far as the second point is The rate of release of the NH 2 -terminal fluorescent product by hfurin at pH 7.0 was measured by an on-line assay as described under "Experimental Procedures."…”

Anti-Müllerian hormone is a gonadal cytokine with two circulating forms and cryptic actions

“…3). Sertoli and granulosa cells express enzymes that cleave proAMH, with the levels of the enzymes and/or their regulators varying during testicular (Nachtigal & Ingraham 1996, Guo et al 2007, Le Magueresse-Battistoni 2007, Uhrin et al 2007) and ovarian (Bae et al 2008) development, the seminiferous cycle (Guo et al 2007, Le Magueresse-Battistoni 2007, Uhrin et al 2007, the ovarian cycle (Bae et al 2008, Wang et al 2014, the stage of ovarian follicular development (Ohnishi et al 2005, Bae et al 2008, Antenos et al 2011) and during pregnancy (Kwok et al 2013). However, the AMH in ovine follicular fluid is predominantly proAMH with little AMH N,C (Campbell et al 2012), suggesting that AMH can be synthesised and released with little or no prior cleavage.…”

“…These include proprotein convertases of the subtilisin/kexin-type 3 (PCSK3) (furin), PCSK5 (PC5 and PC6) and PCSK6 (PACE4) (Nachtigal & Ingraham 1996), which are PCSK. ProAMH is also cleaved by serine proteinases, most notably plasmin (Ragin et al 1992).…”

Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications

“…It may play a role in the cleavage of pro-neurotensin/neuromedin N in human colon cancer cells (Rovere et al 1998). It may be also involved in the process of sexual differentiation, by activating pro-Müllerian substance (Nachtigal & Ingraham 1996). In addition, the transfection of SPC6 in COS cells induces cleavage of the extracellular domains of the receptor protein tyrosine phosphatases (Campan et al 1996).…”