Molekularbiologische Charakterisierung der beiden Untereinheiten des Mega-Hämocyanins der Schnecke Melanoides tuberculata

Bei dem 2010 von unserer Arbeitsgruppe entdeckten Mega-Hämocyanin handelt es sich um einen stark abgewandelten Typ des respiratorischen Proteins Hämocyanin, bestehend aus zwei flankierenden regulären Dekameren und einem zentralen Mega-Dekamer. Diese sind aus zwei immunologisch verschiedenen Untereinheiten mit ~400 bzw. ~550 kDa aufgebaut, die in unserer Arbeitsgruppe bereits proteinbiochemisch charakterisiert wurden. Im Zuge dieser Untersuchungen konnte zudem eine 3D-Rekonstruktion des Oligomers (13,5 MDa) mit einer Auflösung von 13Å erstellt werden. Das Ziel der vorliegenden Arbeit war die Aufklärung der Primärstruktur beider Polypeptide bei der Schnecke Melanoides tuberculata (MtH). Es gelang, die cDNAs der beiden Untereinheiten vollständig zu sequenzieren. Die zu typischen Dekameren assemblierende MtH400-Untereinheit umfasst 3445 Aminosäuren und besitzt eine theoretische Molekularmasse von 390 kDa. Nach dem Signalpeptid von 23 Aminosäuren Länge folgen die für Gastropoden-Hämocyanine typischen funktionellen Einheiten FU-a bis FU-h. Insgesamt verfügt die MtH400-Untereinheit über sechs potentielle N-Glykosylierungsstellen. Die MtH550-Untereinheit, welche mit 10 Kopien das Mega-Dekamer bildet, umfasst 4999 Aminosäuren und besitzt eine theoretische Molekularmasse von 567 kDa. Damit handelt es sich bei dieser Untereinheit um die zweitgrößte jemals bei einem Protein detektierte Polypeptidkette. Die MtH550-Untereinheit besteht aus einem Signalpeptid von 20 Aminosäuren Länge und den typischen Wand-FUs (FU-a bis FU-f). Daran anschließend folgen sechs weitere Varianten der FU-f (FU-f1 bis FU-f6). Die MtH550-Untereinheit verfügt über insgesamt zwölf potentielle N-Glykosylierungsstellen. Anhand der ermittelten Primärstrukturdaten wird klar, dass der auffällig vergrößerte Kragenbereich des Mega-Dekamers aus je 10 Kopien der FU-f1 bis FU-f6 besteht. Die ermittelten Sequenzdaten der beiden MtH-Untereinheiten weisen im Vergleich zu anderen Hämocyanin Sequenzen einige sehr charakteristische Indels sowie unübliche N-Glykosylierungsstellen auf. Es war zudem möglich, anhand einer molekularen Uhr den Entstehungszeitpunkt des Mega-Hämocyanins zu datieren (145 ± 35 MYA). Sowohl die Topologie als auch die berechneten Trennungszeitpunkte des an allen Verzweigungen gut unterstützten Stammbaums stimmen mit den bisher publizierten und auf Hämocyanindaten basierenden molekularen Uhren überein.Previously our group discovered, in cerithioid snails, an unusually complex type of the respiratory protein hemocyanin. This tridecameric mega-hemocyanin is built from two different types of decamer: two flanking decamers of the typical gastropod hemocyanin type, and a central mega-decamer. Each decamer type is built from a specific subunit: ~400 kDa in case of the regular decamer and ~550 kDa in case of the mega-decamer. The two subunit types have been analyzed biochemically, and furthermore, a cryoEM-based 3D-reconstruction of the oligomer (13.5 MDa) with 13Å resolution was provided.rnThe goal of this work was the elucidation of the primary structure from both subunits of the mega-hemocyanin of Melanoides tuberculata. The cDNAs coding for the two subunits could be completely sequenced. The MtH400-subunit comprises 3445 amino acids and has a molecular mass of 390 kDa as predicted from the sequence. It encompasses a signal peptide (23 amino acids) and the eight typical functional units FU-a to FU-h hitherto observed in molluscan hemocyanins. Moreover, it exhibits six potential attachment sites for N-glycans. In contrast, the MtH550-subunit comprises 4999 amino acids and has a predicted molecular mass of 567 kDa. This is the second largest polypeptide ever reported. It encompasses a signal peptide (20 amino acids) and the adjacent canonical functional units FU-a to FU-f which are assumed to form the cylinder wall of the decamer. Instead of the collar-forming functional units FU-g and FU-h as in the MtH400-subunit, the MtH550-subunit possesses C-terminally six variants of FU-f, termed FU-f1 to FU-f6. It exhibits a total of twelve potential N-glycosylation sites, partially at unusual positions. The different functional units of both subunit types show all structural features required for reversible oxygen binding.rnThe major branches of phylogenetic trees calculated from the now available sequence data are well bootstrap-supported and fit the branching pattern of earlier trees of molluscan hemocyanin. Based on a calculated molecular clock, the phylogenetic origin of mega-hemocyanin could be dated back 145 ± 35 million years.r

Morphogenetically-Active Barrier Membrane for Guided Bone Regeneration, Based on Amorphous Polyphosphate

We describe a novel regeneratively-active barrier membrane which consists of a durable electrospun poly(ε-caprolactone) (PCL) net covered with a morphogenetically-active biohybrid material composed of collagen and inorganic polyphosphate (polyP). The patch-like fibrous collagen structures are decorated with small amorphous polyP nanoparticles (50 nm) formed by precipitation of this energy-rich and enzyme-degradable (alkaline phosphatase) polymer in the presence of calcium ions. The fabricated PCL-polyP/collagen hybrid mats are characterized by advantageous biomechanical properties, such as enhanced flexibility and stretchability with almost unaltered tensile strength of the PCL net. The polyP/collagen material promotes the attachment and increases the viability/metabolic activity of human mesenchymal stem cells compared to cells grown on non-coated mats. The gene expression studies revealed that cells, growing onto polyP/collagen coated mats show a significantly (two-fold) higher upregulation of the steady-state-expression of the angiopoietin-2 gene used as an early marker for wound healing than cells cultivated onto non-coated mats. Based on our results we propose that amorphous polyP, stabilized onto a collagen matrix, might be a promising component of functionally-active barrier membranes for guided tissue regeneration in medicine and dentistry

Polyphosphate Reverses the Toxicity of the Quasi-Enzyme Bleomycin on Alveolar Endothelial Lung Cells In Vitro

The anti-cancer antitumor antibiotic bleomycin(s) (BLM) induces athyminic sites in DNA after its activation, a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 µg ml−1 causes a decrease in cell viability (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells (diameter, 20–25 µm) is normalized to ~12 µm after co-incubation of the cells with BLM and polyP. A sequential application of the drugs (BLM for 3 days, followed by an exposure to polyP) does not cause this normalization. During co-incubation of BLM with polyP the gene for the BLM hydrolase is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since polyP inhibits binding of SARS-CoV-2 to cellular ACE2

Development of a 3D simulator for training the mouse in utero electroporation.

In utero electroporation (IUE) requires high-level training in microinjection through the mouse uterine wall into the lateral ventricle of the mouse brain. Training for IUE is currently being performed in live mice as no artificial models allow simulations yet. This study aimed to develop an anatomically realistic 3D printed simulator to train IUE in mice. To this end, we created embryo models containing lateral ventricles. We coupled them to uterus models in six steps: (1) computed tomography imaging, (2) 3D model segmentation, (3) 3D model refinement, (4) mold creation to cast the actual model, (5) 3D mold printing, and (6) mold casting the molds with a mix of soft silicones to ensure the hardness and consistency of the uterus and embryo. The results showed that the simulator assembly successfully recreated the IUE. The compression test did not differ in the mechanical properties of the real embryo or in the required load for uterus displacement. Furthermore, more than 90% of the users approved the simulator as an introduction to IUE and considered that the simulator could help reduce the number of animals for training. Despite current limitations, our 3D simulator enabled a realistic experience for initial approximations to the IUE and is a real alternative for implementing the 3Rs. We are currently working on refining the model

Polyphosphate Reverses the Toxicity of the Quasi-Enzyme Bleomycin on Alveolar Endothelial Lung Cells In Vitro

The anti-cancer antitumor antibiotic bleomycin(s) (BLM) induces athyminic sites in DNA after its activation, a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 µg ml−1 causes a decrease in cell viability (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells (diameter, 20–25 µm) is normalized to ~12 µm after co-incubation of the cells with BLM and polyP. A sequential application of the drugs (BLM for 3 days, followed by an exposure to polyP) does not cause this normalization. During co-incubation of BLM with polyP the gene for the BLM hydrolase is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since polyP inhibits binding of SARS-CoV-2 to cellular ACE2

Inorganic Polymeric Materials for Injured Tissue Repair: Biocatalytic Formation and Exploitation

Two biocatalytically produced inorganic biomaterials show great potential for use in regenerative medicine but also other medical applications: bio-silica and bio-polyphosphate (bio-polyP or polyP). Biosilica is synthesized by a group of enzymes called silicateins, which mediate the formation of amorphous hydrated silica from monomeric precursors. The polymeric silicic acid formed by these enzymes, which have been cloned from various siliceous sponge species, then undergoes a maturation process to form a solid biosilica material. The second biomaterial, polyP, has the extraordinary property that it not only has morphogenetic activity similar to biosilica, i.e., can induce cell differentiation through specific gene expression, but also provides metabolic energy through enzymatic cleavage of its high-energy phosphoanhydride bonds. This reaction is catalyzed by alkaline phosphatase, a ubiquitous enzyme that, in combination with adenylate kinase, forms adenosine triphosphate (ATP) from polyP. This article attempts to highlight the biomedical importance of the inorganic polymeric materials biosilica and polyP as well as the enzymes silicatein and alkaline phosphatase, which are involved in their metabolism or mediate their biological activity

A Novel Biomimetic Approach to Repair Enamel Cracks/Carious Damages and to Reseal Dentinal Tubules by Amorphous Polyphosphate

Based on natural principles, we developed a novel toothpaste, containing morphogenetically active amorphous calcium polyphosphate (polyP) microparticles which are enriched with retinyl acetate (“a-polyP/RA-MP”). The spherical microparticles (average size, 550 ± 120 nm), prepared by co-precipitating soluble Na-polyP with calcium chloride and supplemented with retinyl acetate, were incorporated into a base toothpaste at a final concentration of 1% or 10%. The “a-polyP/RA-MP” ingredient significantly enhanced the stimulatory effect of the toothpaste on the growth of human mesenchymal stem cells (MSC). This increase was paralleled by an upregulation of the MSC marker genes for osteoblast differentiation, collagen type I and alkaline phosphatase. In addition, polyP, applied as Zn-polyP microparticles (“Zn-a-polyP-MP”), showed a distinct inhibitory effect on growth of Streptococcus mutans, in contrast to a toothpaste containing the broad-spectrum antibiotic triclosan that only marginally inhibits this cariogenic bacterium. Moreover, we demonstrate that the “a-polyP/RA-MP”-containing toothpaste efficiently repairs cracks/fissures in the enamel and dental regions and reseals dentinal tubules, already after a five-day treatment (brushing) of teeth as examined by SEM (scanning electron microscopy) and semi-quantitative EDX (energy-dispersive X-ray spectroscopy). The occlusion of the dentin cracks by the microparticles turned out to be stable and resistant against short-time high power sonication. Our results demonstrate that the novel toothpaste prepared here, containing amorphous polyP microparticles enriched with retinyl acetate, is particularly suitable for prevention/repair of (cariogenic) damages of tooth enamel/dentin and for treatment of dental hypersensitivity. While the polyP microparticles function as a sealant for dentinal damages and inducer of remineralization processes, the retinyl acetate acts as a regenerative stimulus for collagen gene expression in cells of the surrounding tissue, the periodontium

Effect of bioglass on growth and biomineralization of SaOS-2 cells in hydrogel after 3D cell bioprinting.

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP • Ca2+-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2 : CaO : P2O5 of 55 : 40 : 5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP • Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP • Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP • Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants

Restoration of Impaired Metabolic Energy Balance (ATP Pool) and Tube Formation Potential of Endothelial Cells under “high glucose”, Diabetic Conditions by the Bioinorganic Polymer Polyphosphate

Micro-vascularization is a fast, energy-dependent process that is compromised by elevated glucose concentrations such as in diabetes mellitus disease. Here, we studied the effect of the physiological bioinorganic polymer, polyphosphate (polyP), on the reduced ATP content and impaired function of endothelial cells cultivated under “high glucose” (35 mM diabetes mellitus conditions) concentrations. This high-energy biopolymer has been shown to provide a source of metabolic energy, stored in its phosphoanhydride bonds. We show that exposure of human umbilical vein endothelial cells (HUVEC cells) to “high glucose” levels results in reduced cell viability, increased apoptotic cell death, and a decline in intracellular ATP level. As a consequence, the ability of HUVEC cells to form tube-like structures in the in vitro cell tube formation assay was almost completely abolished under “high glucose” conditions. Those cells were grown onto a physiological collagen scaffold (collagen/basement membrane extract). We demonstrate that these adverse effects of increased glucose levels can be reversed by administration of polyP to almost normal values. Using Na-polyP, complexed in a stoichiometric (molar) ratio to Ca2+ ions and in the physiological concentration range between 30 and 300 µM, an almost complete restoration of the reduced ATP pool of cells exposed to “high glucose” was found, as well as a normalization of the number of apoptotic cells and energy-dependent tube formation. It is concluded that the adverse effects on endothelial cells caused by the metabolic energy imbalance at elevated glucose concentrations can be counterbalanced by polyP, potentially opening new strategies for treatment of the micro-vascular complications in diabetic patients

Enhancement of Wound Healing in Normal and Diabetic Mice by Topical Application of Amorphous Polyphosphate. Superior Effect of a Host–Guest Composite Material Composed of Collagen (Host) and Polyphosphate (Guest)

The effect of polyphosphate (polyP) microparticles on wound healing was tested both in vitro and in a mice model in vivo. Two approaches were used: pure salts of polyphosphate, fabricated as amorphous microparticles (MPs, consisting of calcium and magnesium salts of polyP, “Ca–polyp-MPs” and “Mg–polyp-MPs”), and host–guest composite particles, prepared from amorphous collagen (host) and polyphosphate (guest), termed “col/polyp-MPs”. Animal experiments with polyP on healing of excisional wounds were performed using both normal mice and diabetic mice. After a healing period of 7 days “Ca–polyp-MP” significantly improved re-epithelialization in normal mice from 31% (control) to 72% (polyP microparticle-treated). Importantly, in diabetic mice, particularly the host–guest particles “col/polyp-MP”, increased the rate of re-epithelialization to ≈40% (control, 23%). In addition, those particles increased the expression of COL-I and COL-III as well as the expression the α-smooth muscle actin and the plasminogen activator inhibitor-1. We propose that “Ca–polyp-MPs”, and particularly the host–guest “col/polyp-MPs” are useful for topical treatment of wounds