Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella.

KEY MESSAGE: Characterization of Physcomitrella 3'UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming. The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular, the good manufacturing practice (GMP)-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features, such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3'UTRs (untranslated regions) of which 7964 transcripts contain at least one intron. Over 91% of 3'UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3'UTRs, 14 terminator candidates were selected and characterized via transient Dual-Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3'UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than tenfold, demonstrating the stronger effect promoters play as compared to terminators. Among selected terminator attributes, the number of polyadenylation sites as well as polyadenylation signals were found to influence terminator performance the most. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

Adaptive evolution of the enigmatic Takakia now facing climate change in Tibet.

The most extreme environments are the most vulnerable to transformation under a rapidly changing climate. These ecosystems harbor some of the most specialized species, which will likely suffer the highest extinction rates. We document the steepest temperature increase (2010-2021) on record at altitudes of above 4,000 m, triggering a decline of the relictual and highly adapted moss Takakia lepidozioides. Its de-novo-sequenced genome with 27,467 protein-coding genes includes distinct adaptations to abiotic stresses and comprises the largest number of fast-evolving genes under positive selection. The uplift of the study site in the last 65 million years has resulted in life-threatening UV-B radiation and drastically reduced temperatures, and we detected several of the molecular adaptations of Takakia to these environmental changes. Surprisingly, specific morphological features likely occurred earlier than 165 mya in much warmer environments. Following nearly 400 million years of evolution and resilience, this species is now facing extinction.

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Structural modelling of human complement FHR1 and two of its synthetic derivatives provides insight into their in-vivo functions.

Human complement is the first line of defence against invading pathogens and is involved in tissue homeostasis. Complement-targeted therapies to treat several diseases caused by a dysregulated complement are highly desirable. Despite huge efforts invested in their development, only very few are currently available, and a deeper understanding of the numerous interactions and complement regulation mechanisms is indispensable. Two important complement regulators are human Factor H (FH) and Factor H-related protein 1 (FHR1). MFHR1 and MFHR13, two promising therapeutic candidates based on these regulators, combine the dimerization and C5-regulatory domains of FHR1 with the central C3-regulatory and cell surface-recognition domains of FH. Here, we used AlphaFold2 to model the structure of these two synthetic regulators. Moreover, we used AlphaFold-Multimer (AFM) to study possible interactions of C3 fragments and membrane attack complex (MAC) components C5, C7 and C9 in complex with FHR1, MFHR1, MFHR13 as well as the best-known MAC regulators vitronectin (Vn), clusterin and CD59, whose experimental structures remain undetermined. AFM successfully predicted the binding interfaces of FHR1 and the synthetic regulators with C3 fragments and suggested binding to C3. The models revealed structural differences in binding to these ligands through different interfaces. Additionally, AFM predictions of Vn, clusterin or CD59 with C7 or C9 agreed with previously published experimental results. Because the role of FHR1 as MAC regulator has been controversial, we analysed possible interactions with C5, C7 and C9. AFM predicted interactions of FHR1 with proteins of the terminal complement complex (TCC) as indicated by experimental observations, and located the interfaces in FHR11-2 and FHR14-5. According to AFM prediction, FHR1 might partially block the C3b binding site in C5, inhibiting C5 activation, and block C5b-7 complex formation and C9 polymerization, with similar mechanisms of action as clusterin and vitronectin. Here, we generate hypotheses and give the basis for the design of rational approaches to understand the molecular mechanism of MAC inhibition, which will facilitate the development of further complement therapeutics.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention.

The auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

Process Engineering of Biopharmaceutical Production in Moss Bioreactors via Model-Based Description and Evaluation of Phytohormone Impact.

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

Unexpected Arabinosylation after Humanization of Plant Protein N-Glycosylation.

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with ß1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human ß1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric ß1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human ß1,4-galactosyltransferase. Stable FTGT expression led to the desired ß1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of ß1,4-galactosylated N-glycans was reported earlier, e.g., on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

A synthetic protein as efficient multitarget regulator against complement over-activation.

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g., COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may be alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH, including SCR 13. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as therapeutic for complement-associated diseases.

O-methylated N-glycans Distinguish Mosses from Vascular Plants.

In the animal kingdom, a stunning variety of N-glycan structures have emerged with phylogenetic specificities of various kinds. In the plant kingdom, however, N-glycosylation appears to be strictly conservative and uniform. From mosses to all kinds of gymno- and angiosperms, land plants mainly express structures with the common pentasaccharide core substituted with xylose, core α1,3-fucose, maybe terminal GlcNAc residues and Lewis A determinants. In contrast, green algae biosynthesise unique and unusual N-glycan structures with uncommon monosaccharides, a plethora of different structures and various kinds of O-methylation. Mosses, a group of plants that are separated by at least 400 million years of evolution from vascular plants, have hitherto been seen as harbouring an N-glycosylation machinery identical to that of vascular plants. To challenge this view, we analysed the N-glycomes of several moss species using MALDI-TOF/TOF, PGC-MS/MS and GC-MS. While all species contained the plant-typical heptasaccharide with no, one or two terminal GlcNAc residues (MMXF, MGnXF and GnGnXF, respectively), many species exhibited MS signals with 14.02 Da increments as characteristic for O-methylation. Throughout all analysed moss N-glycans, the level of methylation differed strongly even within the same family. In some species, methylated glycans dominated, while others had no methylation at all. GC-MS revealed the main glycan from Funaria hygrometrica to contain 2,6-O-methylated terminal mannose. Some mosses additionally presented very large, likewise methylated complex-type N-glycans. This first finding of the methylation of N-glycans in land plants mirrors the presumable phylogenetic relation of mosses to green algae, where the O-methylation of mannose and many other monosaccharides is a common trait.

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella.

KEY MESSAGE: In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA-DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

Expression of a human cDNA in moss results in spliced mRNAs and fragmentary protein isoforms.

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.

Axenic in vitro cultivation of 19 peat moss (Sphagnum L.) species as a resource for basic biology, biotechnology, and paludiculture.

Sphagnum farming can substitute peat with renewable biomass and thus help mitigate climate change. Large volumes of the required founder material can only be supplied sustainably by axenic cultivation in bioreactors. We established axenic in vitro cultures from sporophytes of 19 Sphagnum species collected in Austria, Germany, Latvia, the Netherlands, Russia, and Sweden: S. angustifolium, S. balticum, S. capillifolium, S. centrale, S. compactum, S. cuspidatum, S. fallax, S. fimbriatum, S. fuscum, S. lindbergii, S. medium/divinum, S. palustre, S. papillosum, S. rubellum, S. russowii, S. squarrosum, S. subnitens, S. subfulvum and S. warnstorfii. These species cover five of the six European Sphagnum subgenera; namely, Acutifolia, Cuspidata, Rigida, Sphagnum and Squarrosa. Their growth was measured in suspension cultures, whereas their ploidy was determined by flow cytometry and compared with the genome size of Physcomitrella patens. We identified haploid and diploid Sphagnum species, found that their cells are predominantly arrested in the G1 phase of the cell cycle, and did not find a correlation between plant productivity and ploidy. DNA barcoding was achieved by sequencing introns of the BRK1 genes. With this collection, high-quality founder material for diverse large-scale applications, but also for basic Sphagnum research, is available from the International Moss Stock Center.

Mosses in biotechnology.

Biotechnological exploitation of mosses has several aspects, for example, the use of moss extracts or the whole plant for diverse industrial applications as well as their employment as production platforms for valuable metabolites or pharmaceutical proteins, especially using the genetically and developmentally best-characterised model moss Physcomitrella patens. Whole moss plants, in particular peat mosses (Sphagnum spec.), are useful for environmental approaches, biomonitoring of environmental pollution and CO2-neutral 'farming' on rewetted bogs to combat climate change. In addition, the lifestyle of mosses suggests the evolution of genes necessary to cope with biotic and abiotic stress situations, which could be applied to crop plants, and their structural features bear an inspiring potential for biomimetics approaches.

Stable Protein Sialylation in Physcomitrella.

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the ß1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of ß1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human ß1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous ß1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

Recombinant Production of MFHR1, A Novel Synthetic Multitarget Complement Inhibitor, in Moss Bioreactors.

The human complement system is an important part of the immune system responsible for lysis and elimination of invading microorganisms and apoptotic body cells. Improper activation of the system due to deficiency, mutations, or autoantibodies of complement regulators, mainly factor H (FH) and FH-related proteins (FHRs), causes severe kidney and eye diseases. However, there is no recombinant FH therapeutic available on the market. The first successful recombinant production of FH was accomplished with the moss bioreactor, Physcomitrella patens. Recently, a synthetic regulator, MFHR1, was designed to generate a multitarget complement inhibitor that combines the activities of FH and the FH-related protein 1 (FHR1). The potential of MFHR1 was demonstrated in a proof-of-concept study with transiently transfected insect cells. Here, we present the stable production of recombinant glyco-engineered MFHR1 in the moss bioreactor. The key features of this system are precise genome engineering via homologous recombination, Good Manufacturing Practice-compliant production in photobioreactors, high batch-to-batch reproducibility, and product stability. Several potential biopharmaceuticals are being produced in this system. In some cases, these are even biobetters, i.e., the recombinant proteins produced in moss have a superior quality compared to their counterparts from mammalian systems as for example moss-made aGal, which successfully passed phase I clinical trials. Via mass spectrometry-based analysis of moss-produced MFHR1, we now prove the correct synthesis and modification of this glycoprotein with predominantly complex-type N-glycan attachment. Moss-produced MFHR1 exhibits cofactor and decay acceleration activities comparable to FH, and its mechanism of action on multiple levels within the alternative pathway of complement activation led to a strong inhibitory activity on the whole alternative pathway, which was higher than with the physiological regulator FH.

Critical Evaluation of Strategies for the Production of Blood Coagulation Factors in Plant-Based Systems.

The use of plants as production platforms for pharmaceutical proteins has been on the rise for the past two decades. The first marketed plant-made pharmaceutical, taliglucerase alfa against Gaucher's disease produced in carrot cells by Pfizer/Protalix Biotherapeutics, was approved by the US Food and Drug Administration (FDA) in 2012. The advantages of plant systems are low cost and highly scalable biomass production compared to the fermentation systems, safety compared with other expression systems, as plant-based systems do not produce endotoxins, and the ability to perform complex eukaryotic post-translational modifications, e.g., N-glycosylation that can be further engineered to achieve humanized N-glycan structures. Although bleeding disorders affect only a small portion of the world population, costs of clotting factor concentrates impose a high financial burden on patients and healthcare systems. The majority of patients, ∼75% in the case of hemophilia, have no access to an adequate treatment. The necessity of large-scale and less expensive production of human blood coagulation factors, particularly factors associated with rare bleeding disorders, may be an important area for plant-based systems, as coagulation factors do not fit into the industry-favored production models. In this review, we explore previous studies on recombinant production of coagulation Factor II, VIII, IX, and XIII in different plant species. Production of bioactive FII and FIX in plants was not achieved yet due to complex post-translational modifications, including vitamin K-dependent γ-carboxylation and propeptide removal. Although plant-made FVIII and FXIII showed specific activities, there are no follow-up studies like pre-clinical/clinical trials. Significant progress has been achieved in oral delivery of bioencapsulated FVIII and FIX to induce immune tolerance in murine models of hemophilia A and B, resp. Potential strategies to overcome bottlenecks in the production systems are also addressed in this review.

Host Cell Proteome of Physcomitrella patens Harbors Proteases and Protease Inhibitors under Bioproduction Conditions.

Host cell proteins are inevitable contaminants of biopharmaceuticals. Here, we performed detailed analyses of the host cell proteome of moss ( Physcomitrella patens) bioreactor supernatants using mass spectrometry and subsequent bioinformatics analysis. Distinguishing between the apparent secretome and intracellular contaminants, a complex extracellular proteolytic network including subtilisin-like proteases, metallo-proteases, and aspartic proteases was identified. Knockout of a subtilisin-like protease affected the overall extracellular proteolytic activity. Besides proteases, also secreted protease-inhibiting proteins such as serpins were identified. Further, we confirmed predicted cleavage sites of 40 endogenous signal peptides employing an N-terminomics approach. The present data provide novel aspects to optimize both product stability of recombinant biopharmaceuticals as well as their maturation along the secretory pathway. Data are available via ProteomeXchange with identifier PXD009517.

RecQ Helicases Function in Development, DNA Repair, and Gene Targeting in Physcomitrella patens.

RecQ DNA helicases are genome surveillance proteins found in all kingdoms of life. They are characterized best in humans, as mutations in RecQ genes lead to developmental abnormalities and diseases. To better understand RecQ functions in plants we concentrated on Arabidopsis thaliana and Physcomitrella patens, the model species predominantly used for studies on DNA repair and gene targeting. Phylogenetic analysis of the six P. patens RecQ genes revealed their orthologs in humans and plants. Because Arabidopsis and P. patens differ in their RecQ4 and RecQ6 genes, reporter and deletion moss mutants were generated and gene functions studied in reciprocal cross-species and cross-kingdom approaches. Both proteins can be found in meristematic moss tissues, although at low levels and with distinct expression patterns. PpRecQ4 is involved in embryogenesis and in subsequent development as demonstrated by sterility of ΔPpRecQ4 mutants and by morphological aberrations. Additionally, ΔPpRecQ4 displays an increased sensitivity to DNA damages and an increased rate of gene targeting. Therefore, we conclude that PpRecQ4 acts as a repressor of recombination. In contrast, PpRecQ6 is not obviously important for moss development or DNA repair but does function as a potent enhancer of gene targeting.

The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential.

The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.