Phylogenomic analyses reveal the evolutionary origin of the inhibin alpha-subunit, a unique TGFbeta superfamily antagonist

Abstract

Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.

Figure 1. Plausible models of evolution for the inhibin/activin gene family.

A) Simplified species tree for animals based upon recent large-scale analyses , , . “WGD” indicates the position of the whole genome duplications uniting vertebrates and letters indicate alternative models for the origin of the α-subunit (the letters used correspond to the parts of this figure). B) Cladogram showing the expected inhibin/activin phylogeny given the “early vertebrate duplication” model. Two rounds of WGD have been suggested to characterize vertebrates , , and we show the only topology consistent both with two rounds of WGD and a clade containing INHBA-INHBB (the latter clade is strongly-supported in this study and in other studies; , . C) Cladogram showing the expected phylogeny of inhibin/activin genes given the “deuterostome duplication” model, which places the α-subunit origin before the early vertebrate WGDs. Several versions of this model are possible (i.e., the α-subunit origin could predate the divergence of vertebrates from urochordates, cephalochordates, or even echinoderms). D) Cladogram showing the expected inhibin/activin phylogeny given the “early animal duplication” model, which places the α-subunit origin before the divergence of deuterosomes and protostomes. The “Dawdle orthologue” hypothesis is a version of this model. Additional duplications may have occurred in any of these models. Biased estimation of the gene tree or sampling variance may cause estimates of the gene tree to deviate from any of these idealized model trees.

Figure 2. Phylogenomic analyses revealed a complex series of indels that correlated with major events in the evolution of the inhibin α-subunit.

A) Alignment of mature inhibin/activin proteins showing expanded sampling within the vertebrates for the α-subunit. The alignments were optimized based upon the highly conserved cysteine residues and the highly conserved W-X-X-W motif and R-X-X-R proconvertase enzyme cleavage site (bold). N-terminal region and wrist region were highlighted with grey shade. B) Cladogram showing evolutionary relationships among animals with annotated genome sequences available using a topology based upon a consensus of recent analyses , , . The two major clades of bilaterian animals (deuterostomes and protostomes) are highlighted. Numbers of proteins that exhibit clear homology to inhibin/activin queries in BLASTP searches are shown to the right, with the number of those proteins that have a human inhibin/activin α- or β-subunit as their top hit when used as a query in BLASTP searches of human proteins indicated in parentheses. Thus, numbers in parentheses reflect the number of proteins that are candidates for inhibin/activin α- or β-subunits using a bidirectional BLAST criterion. The likely origins of α- and β-subunits based upon the phylogenomic analyses reported here are indicated using the relevant Greek letters, and the timing of the whole genome duplications uniting vertebrates are indicated as “WGD”. The branch at the base of the tree is hatched because the position of the root is unclear , , . C) Schematic of the ML estimates the phylogeny for inhibin/activin proteins, emphasizing the occurrence of indels in the mature protein region during the evolution of the gene family. Support for specific groups is indicated as the percentage of 100 bootstrap replicates, with only values ≥50% indicated. The starlet sea anemone (Nematostella) activin homolog is the sister of the lancelet (Branchiostoma) β-like protein and is indicated using a light line since it is probably placed incorrectly (note that bootstrap support is limited). The dashed arrow indicates the likely position of the sea anemone activin; this position is more likely because it minimizes the number of gene duplications and losses given the likely organismal phylogeny shown in B). A detailed version of this phylogeny is provided in Fig. S3.

Figure 3. Cloning and expression of engineered inhibin and activin mutants and their wild type molecules.

Schematic representations of wild type and mutant inhibin/activin molecules along with immunoblots demonstrating their presence in conditioned media from stably transfected cells. The construct transfected is marked above each lane and the product bands are indicated on the side with lower case letters or numbers or Greece letters that correspond to the schematic representation on the left. N-linked glycosylation site is denoted by the ψ symbol. Details on the design and nomenclature of all the mutants described herein are provided in Fig. S4 and Table S3. A) Left, schematic representations of dimeric and monomeric forms of wild type activin (βA/βA), activin chimera mutants (βA^ext+/βA^ext+ or βA^PWR+/βA^PWR+) and activin deletion mutant (βA^WHD/βA^WHD). Right, immunoblot of wild type activin and its chimera and deletion mutants using anti-βA-subunit polyclonal antibody under non-reducing (NR) and reducing (R) conditions. B) Left, schematic representations of wild type chicken inhibin (α^Chwt/βA) as well as dimeric and monomeric forms of chicken free α-subunit (α^Chwt/α^Chwt, α^Chwt and Pro-α^Chwt). Middle, immunoblot of wild type chicken free α-subunit and chicken inhibin using anti-human-α-subunit PO23 monoclonal antibody under non-reducing (NR) or reducing (R) conditions. Far Right, non-reducing SDS-PAGE autoradiograph showing the immunoprecipitation (using anti-α-subunit monoclonal antibody PO23) of [³⁵S]-cysteine-labeled chicken free α-subunit and chicken inhibin A. Controls for the immunoprecipitation experiment are shown in Fig. S5A. C) Left, schematic representations of human wild type inhibin A (α^Hwt/βA), chicken wild type inhibin (α^Chwt/βA), and three deletion mutants of the human α-subunit (α^Hext−/βA, α^HPRW−/βA and α^Hext-PRW−/βA). Right, immunoblot of wild type human and chicken inhibin A and the human α-subunit deletion mutants using anti-α-subunit PO23 monoclonal antibody under non-reducing (NR) condition. Subsequent immunoblots detected with either anti-α-subunit monoclonal antibody PO23 or anti-β_A-subunit polyclonal antibody under reducing conditions are shown in Fig. S6.

Figure 4. Equilibrium binding of activin and inhibin mutants to ActRIIB using competitive ELISA and immunoprecipitation studies.

A) Conditioned media from βA/βA, βA^ext+/βA^ext+, and βA^WHD/βA^WHD expressing cells were quantified and used to compete with biotinylated activin A for binding to ActRIIB. The IC₅₀ for the βA/βA, βA^ext+/βA^ext+ and βA^WHD/βA^WHD competition binding curves were 4.14±31.61 nM, 5.99±0.98 nM, 47.6±14.3 nM, respectively, representing a significant difference between all groups as determined by sigmoidal dose-response (variable slope) curve, followed by an F-test (p<0.05). The standard curve for biotinylated activin A binding to the ActRIIB (EC₅₀ = 1.1 nM) is shown in Fig. S7A. B) Top, densitometric analysis of immunoblots (bottom) from three independent experiments, Density was normalized to COS7 cells transfected with empty vector (pcDNA3) and treated with human wild type inhibin A. Asterisks represent statistically significant differences using unpaired t-test (p<0.05). Bottom, the immunoblots of the immunoprecipitation that use monoclonal anti-HA pulldown for lysates from COS-7 cells transfected with ActRIIB-HA and either full-length betaglycan or empty vector (pcDNA3). Prior to cell lysis, the cells were treated with inhibin deletion mutants, wild type human inhibin A, chicken inhibin A or activin A culture media for 2 hours. The western-blots are detected by anti-β_A-subunit polyclonal antibody. Controls for the immunoprecipitation studies are shown in Fig. S8. C) Conditioned media from α^Hwt/βA, α^CHwt/βA, α^Hext−/βA and α^Hwr−/βA expressing cells were quantified and used to compete with biotinylated inhibin A for binding to ActRIIB. The IC₅₀ for the α^Hwt/βA, α^CHwt/βA, α^Hwr−/βA and α^Hext−/βA competition binding curves were 9.01±3.72 nM, 5.55±1.85 nM, 2.96±1.23 nM and 127.9±17.80 nM, respectively, representing a significant difference between all groups as determined by sigmoidal dose-response (variable slope) curve, followed by F-test (P<0.05). The standard curve for biotinylated inhibin A binding to the ActRIIB (EC₅₀ = 2.73 nM) is shown in Fig. S7B. The graphs shown in (A) and (C) represent the results of more than three independent experiments.

Figure 5. Schematic diagrams illustrating the proposed model for the evolution of the inhibin α-subunit.

A) Indels that have had a major impact upon α-subunit function are indicated on the deuterostome tree. Two major indel changes (the N-terminal insertion and wrist helix region deletion and the proline-rich wrist region insertion [2]) occurred at distinct times in evolution. B) Schematic representation of the evolution of inhibin α-subunit. The graph includes the forward engineered mutants used in our experiment that are representative of α-like transient forms. Non-mammalian α-subunit structure is represented by the chicken α orthologue, and mammalian α-subunit structure is diagramed by the human α orthologue. C) Evolution from inhibin β-subunit to inhibin α-subunit involves the loss of the ability to homodimerize. Concurrent with this evolution was a change in the bioactivity from agonist to strong antagonist that is betaglycan independent, and later to weak antagonist that requires betaglycan for maximal antagonistic function. The indel in the evolution of inhibin α-subunit strongly affects inhibin function.