Biology subjects

Bohlender, L. L.

Publications and source records attributed to Bohlender, L. L..

7 recordsLinked to original sources

bioRxiv2025

Production of prolyl-hydroxylated human type III collagen in Physcomitrella photobioreactors

Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO2 supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. Key messageScalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals.

plant biology↗

bioRxiv2024

Recombinant production of spider silk protein in Physcomitrella photobioreactors

Spider dragline silk stands out as a remarkable biomaterial, representing one of natures toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, (LhMaSp1) in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N- and C-terminal domains (NTD and CTD) of LhMaSp1 protein in moss cells. NTD and CTD are necessary domains for protein assembly in spider silk. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. Key messageWe report the first successful plant-produced recombinant spider silk key protein component containing both the N- and the C-terminal domain.

plant biology↗

bioRxiv2024

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are undesired on plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95% of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs. HighlightsMosses, such as Physcomitrella, encode six prolyl 4-hydroxylases in four subfamilies Computational analysis reveals specific environment for proline hydroxylation AlphaFold structure models and mass spectrometry reveal features of modification Quantitative proteomics and mutant analysis identify relevant modifying enzyme Modelling suggests relevant amino acids in active centre of the modifying enzyme

plant biology↗

bioRxiv2024

Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins

1.Bacterial cell division and plant chloroplast division require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

plant biology↗

bioRxiv2021

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 {beta}1,4-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 {beta}1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric {beta}1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss 1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human {beta}1,4-galactosyltransferase. Stable FTGT expression led to the desired {beta}1,4-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 -linked arabinoses. Since a pentosylation of {beta}1,4-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.

bioengineering↗

bioRxiv2021

The synthetic protein MFHR13 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 present 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. MFHR13 includes the FH variant I62, which we characterized to induce improved C3b binding and cofactor activity compared to the variant V62. After comparative protein structure modelling, we introduced the SCR FH13, which includes an N-glycosylation site for higher protein stability. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It shows an enhanced heparin binding and protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, it also blocks the terminal pathway of complement activation and 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 a therapeutic for complement-associated diseases.

immunology↗

bioRxiv2020

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. Physcomitrella (Physcomitrium patens, 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 {beta}1,2-xylosyltransferase and 1,3-fucosyltransferase genes ({Delta}xt/ft). Furthermore, P. patens 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 P. patens. The sialylation ability was achieved in a {Delta}xt/ft moss line by stable expression of six mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for synthesis, activation, transport and transfer of sialic acid. Production of free and (CMP)-activated sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human {beta}1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous {beta}1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

bioengineering↗