Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Wilson, Cheryl A.; Clemente, Nathalie di; Ehrenfels, Christian W.; Pepinsky, R. Blake; Josso, Nathalie; Vigier, B.; Cate, Richard L.

doi:10.1210/mend.7.2.8469238

Cited by 63 publications

(59 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A small proportion of cleaved MIS was detected in these protein preparations, leading to the subsequent proposal that proteolytic processing of MIS does occur, but at its target organ and not at its site of secretion (3,4). Previous to our work, others have shown that purified recombinant MIS is correctly cleaved at the monoarginyl (P1) site after incubation with the serine protease plasmin (3,28), suggesting that proteolytic bioactivation of MIS may occur at its target site, possibly through the action of the plasminogen activator system. Since the primary targets of the plasminogen activator system reside in the extracellular environment, this system would not contribute to the intracellular processing of MIS that we observe.…”

Section: Resultsmentioning

confidence: 71%

“…Cate and coworkers have shown that the COOH terminus of MIS, by itself, is fully active. Interestingly the NH2-terminal prodomain of MIS, which is not inherently bioactive (28,35), enhances activity significantly, leading to the speculation that the proregion contributes to protein stability and/or participates in the presentation of the mature MIS protein to its cell surface receptor. We find, as others, that the NH2-and COOHterminal MIS fragments remain associated noncovalently after secretion.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Nachtigal

Ingraham

1996

Proc. Natl. Acad. Sci. U.S.A.

125

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

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

Nachtigal

Ingraham

1996

Proc. Natl. Acad. Sci. U.S.A.

125

View full text Add to dashboard Cite

show abstract

“…The Nterminal domain, apart from being probably required for folding and secretion of the C-terminal domain, has no own biological activity, and can even, as in the case of TGF-β, inactivate the mature protein (Massagué, 1990). In contrast, the bioactivity of human AMH has been demonstrated to be dramatically increased by association with its N-terminal domain in vitro (Wilson et al, 1993). It is therefore tempting to speculate that sb-AMH variants comprising different N-terminal domains (as in the case of sb-AMHexonII/− 99) or those whose C-terminal domain is not translated (as in the case of sb-AMHexonVII/+ 5) might exert distinct effects on the bioactivity of sb-AMH.…”

Section: Discussionmentioning

confidence: 99%

Anti-Müllerian hormone (AMH/AMH) in the European sea bass: Its gene structure, regulatory elements, and the expression of alternatively-spliced isoforms

Halm

Rocha

Miura

et al. 2007

Gene

View full text Add to dashboard Cite

In mammals, a multitude of studies have shown that anti-Müllerian hormone (AMH/AMH), apart from inducing Müllerian duct regression during male sexual differentiation, exerts inhibitory effects on male and female gonadal steroidogenesis and differentiation. However, in lower vertebrates like teleost fish, the function of AMH/AMH has been far less explored. As a first step to unravel its potential role in reproduction in teleost fish, we isolated and characterised the AMH gene in the European sea bass (sb), Dicentrachus labrax, determined putative regulatory elements of its 5'-flanking region, and analysed its gene expression and those of alternatively-spliced transcripts. The characterisation of sb-AMH revealed distinct features that distinguishes it from mammalian and bird AMH, suggesting a high rate of diversification of AMH during vertebrate evolution. It contained 7 exons that were divided by 6 introns, of which the last intron (intron vi) was localised only a few nucleotides upstream of the putative peptide cleavage site. The guanine and cytosine content of the open reading frame (ORF) was 52.7% and thus notably lower than that of bird and mammalian AMH. Sb-AMH cDNA was 2045 base pairs (bp) long, containing an ORF of 1599 bp encoding 533 amino acids. Deduced amino acid similarities of the conserved, carboxyterminal domain were highest with AMH in Japanese flounder (84.2%) and lowest with chicken AMH (45.5%). In the proximal promoter sequence of sb-AMH, a steroidogenic factor-1 (SF-1) binding site was present; however other regulatory sequences essential for transcriptional activation of AMH in mammals were absent. Likewise, there was no sequence homology to an SF3A2 sequence within the first 3200 bp upstream of the sb-AMH translation start site. Gene expression of sb-AMH and of alternatively-spliced sb-AMH transcripts were analysed in male and female juvenile and adult gonads as well as in somatic tissues of juvenile males. sb-AMH expression was highest in juvenile testis, but still remarkably high in juvenile ovaries and adult testis, as well as in brain, pituitary, and heart of juvenile male sea bass. Apart from adult ovary, levels of alternatively-spliced sb-AMHexonII/-99 were marginal in comparison with sb-AMH. In contrast, the transcript variant sb-AMHexonVII/+5 was expressed to a similar extent as sb-AMH in all tissues examined. The results of this work have provided the basis for future studies concerning the regulation and function of AMH/AMH in this species.

show abstract

“…After proteolytic processing in the trans-Golgi network by members of the family of subtilisin-like prohormone convertases (furins; see review by Nakayama (62)), two representatives of the TGF-␤ superfamily, TGF-␤1 (63,64) and Mü llerian inhibiting substance (65), are secreted from cultured cells and embryonic testes, respectively, as non-covalent complexes. The prodomains of TGF-␤1 (66,67) and Mü llerian inhibiting substance (68) have also been shown to reassociate with their respective signaling ligands in vitro, demonstrating that stable, non-covalent complexes between the N-and Cterminal domains form both in vitro and in vivo. The interface between the two domains almost certainly includes, and thus renders solvent-inaccessible, the hydrophobic surface patches of the folded dimer, since the largest or the second largest patch forms the intersubunit interface in 90% of all multimeric proteins (69).…”

Section: Discussionmentioning

confidence: 99%

Biochemical and Biophysical Characterization of RefoldedDrosophila DPP, a Homolog of Bone Morphogenetic Proteins 2 and 4

Groppe

Rumpel

Economides

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15°C). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coliexpressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250 -400 amino acids) N-terminal prodomains of transforming growth factor-␤ superfamily members, shown to act as intramolecular chaperones in vivo.The decapentaplegic locus (dpp) encodes a signaling ligand homologous to human bone morphogenetic proteins BMP-2 1 and -4 (1, 2) and has been the object of intense study due to its importance in many fundamental processes during Drosophila development. The mature DPP ligand is secreted as a disulfidelinked homodimer of the C-terminal domain of a large proprotein precursor and has been shown to act as a morphogen required for the establishment of the dorsoventral axis of the embryo (3) and to impart positional information over long range to specify cell fate along the anterior/posterior axis of the limbs (4, 5). DPP signaling also occurs at short range between germ layers of the developing gut (6 -8) and is required to direct the dorsal and ventral migration of tracheal cells during embryogenesis (9) and to act as a relay signal triggering epithelial cell shape changes during the process of dorsal closure (10). In contrast to the developmental roles of DPP signaling, which have been the focus of extensive genetic studies, little is known about the folding, modification, and processing of the DPP proprotein, its distribution and state extracellularly, or the biochemical and biophysical properties of the mature homodimer and specific aspects of its structure.Characterization of the protein products of the dpp locus has been hindered by...

show abstract

Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily.

Cited by 63 publications

References 18 publications

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

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

Anti-Müllerian hormone (AMH/AMH) in the European sea bass: Its gene structure, regulatory elements, and the expression of alternatively-spliced isoforms

Biochemical and Biophysical Characterization of RefoldedDrosophila DPP, a Homolog of Bone Morphogenetic Proteins 2 and 4

Contact Info

Product

Resources

About