Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell–like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.
SummaryApplication of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48 ⁄ 45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plantproduced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48 ⁄ 45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.
Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.
Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.
In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.
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