Specific localization of inorganic polyphosphate (poly P) in fungal cell walls by selective extraction and immunohistochemistry

Werner, Thomas P.; Amrhein, Nikolaus; Freimoser, Florian M.

doi:10.1016/j.fgb.2007.01.008

Cited by 49 publications

(54 citation statements)

References 39 publications

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“…We then compared the cofactor activity of polyP versus dextran sulfate and found that FXI activation by ␣-thrombin in the presence of 4M polyP (255mer) was significantly greater than that obtained in the presence of 1 g/mL dextran sulfate ( Figure 1C). The isolated polyP-binding domain of EcPPXc has been used previously as a specific probe for localizing polyP in yeast cell walls, 25,29 so we examined its ability to specifically inhibit the cofactor function of polyP. The inclusion of 30 g/mL EcPPXc profoundly inhibited FXI activation by ␣-thrombin in the presence of polyP, but had no effect on FXI activation by ␣-thrombin in the presence of dextran sulfate ( Figure 1C).…”

Section: Results Polyp Enhances Fxi Activation By Thrombinmentioning

confidence: 97%

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Polyphosphate is a cofactor for the activation of factor XI by thrombin

Choi

Smith

Morrissey

2011

Blood

215

282

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Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by ␣-thrombin, ␤-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis. (Blood. 2011;118(26):6963-6970) IntroductionIn the original cascade or waterfall model of coagulation, 1 factor XI (FXI) is activated by factor XIIa (FXIIa), a member of the contact pathway of blood clotting. Patients with severe FXI deficiency may exhibit bleeding tendencies, 2,3 especially postoperative or posttraumatic bleeding in tissues with robust fibrinolytic activity. [4][5][6] Conversely, individuals with severe deficiencies in FXII, high-molecularweight kininogen, or prekallikrein do not exhibit bleeding diatheses at all, indicating that the proteins responsible for triggering the classic contact pathway of blood clotting are completely dispensable for hemostasis. 7 Thus, in normal hemostasis, FXI must be activated in vivo by a protease other than FXIIa. A solution to this conundrum was proposed in 1991 by Naito and Fujikawa 8 and by Gailani and Broze,9 who reported that thrombin up-regulates its own generation by feeding back to activate FXI, leading to a "revised model of coagulation." [9][10][11] More recently, Matafonov et al identified that ␤-thrombin and ␥-thrombin, proteolyzed derivatives of ␣-thrombin, also can activate FXI in plasma. 12 The proposal that FXI activation by thrombin plays a significant role in blood clotting in vivo is somewhat controversial. [13][14][15] In solution, the rates both of FXI activation by thrombin and of FXI autoactivation are slow but are markedly enhanced in the presence of polyanions, 8,9,16,17 although most studies have used nonphysiologic cofactors such as dextran sulfate or high concentrations of sulfatides. The relevant physiologic cofactors for FXI activation by thrombin in plasma, if any, have yet to be definitely determined.Polyphosphate (polyP), a linear polymer of inorganic phosphate residues, accumulates in a variety of microorganisms 18 and is secreted by activated human platelets. 19 We recently showed that polyP is a potent modulator of the human blood clotting system, acting at 3 points in...

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Section: Results Polyp Enhances Fxi Activation By Thrombinmentioning

confidence: 97%

“…EcPPXc, the recombinant polyP-binding domain of Escherichia coli exopolyphosphatase fused to maltose-binding protein and a His 6 tag, was produced as described previously. 25 Recombinant Saccharomyces cerevisiae exopolyphosphatase fused to a His 6 tag (rPPX1) was produced as described previously. 26 …”

Section: Methodsmentioning

confidence: 99%

Polyphosphate is a cofactor for the activation of factor XI by thrombin

Choi

Smith

Morrissey

2011

Blood

215

282

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“…The IR spectrum of M . circinelloides biomass before extraction (BE) showed high intensities at 875 and 1260 cm -1 related to P-O and P = O stretching which could be explained by the presence of polyphosphates in the cell wall of Mucor species [67–69] (Fig 1A). These peaks disappear in the spectrum of the biomass after Lewis extraction, indicating that the HCl employed during extraction hydrolyzes the polyphosphates located between the cell wall and the cytoplasmic membrane.…”

Section: Resultsmentioning

confidence: 99%

FTIR Spectroscopy for Evaluation and Monitoring of Lipid Extraction Efficiency for Oleaginous Fungi

et al. 2017

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To assess whether Fourier Transform Infrared (FTIR) spectroscopy could be used to evaluate and monitor lipid extraction processes, the extraction methods of Folch, Bligh and Lewis were used. Biomass of the oleaginous fungi Mucor circinelloides and Mortierella alpina were employed as lipid-rich material for the lipid extraction. The presence of lipids was determined by recording infrared spectra of all components in the lipid extraction procedure, such as the biomass before and after extraction, the water and extract phases. Infrared spectra revealed the incomplete extraction after all three extraction methods applied to M.circinelloides and it was shown that mechanical disruption using bead beating and HCl treatment were necessary to complete the extraction in this species. FTIR spectroscopy was used to identify components, such as polyphosphates, that may have negatively affected the extraction process and resulted in differences in extraction efficiency between M.circinelloides and M.alpina. Residual lipids could not be detected in the infrared spectra of M.alpina biomass after extraction using the Folch and Lewis methods, indicating their complete lipid extraction in this species. Bligh extraction underestimated the fatty acid content of both M.circinelloides and M.alpina biomass and an increase in the initial solvent-to-sample ratio (from 3:1 to 20:1) was needed to achieve complete extraction and a lipid-free IR spectrum. In accordance with previous studies, the gravimetric lipid yield was shown to overestimate the potential of the SCO producers and FAME quantification in GC-FID was found to be the best-suited method for lipid quantification. We conclude that FTIR spectroscopy can serve as a tool for evaluating the lipid extraction efficiency, in addition to identifying components that may affect lipid extraction processes.

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“…In Saccharomyces cerevisiae , polyP is accumulated in both the extracellular space and the vacuoles [68]. PolyP synthesis is connected with the vacuolar transporter chaperone complex (VTC).…”

Section: Ppks As Energetic Enzymesmentioning

confidence: 99%

Polyphosphate - an ancient energy source and active metabolic regulator

2011

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There are a several molecules on Earth that effectively store energy within their covalent bonds, and one of these energy-rich molecules is polyphosphate. In microbial cells, polyphosphate granules are synthesised for both energy and phosphate storage and are degraded to produce nucleotide triphosphate or phosphate. Energy released from these energetic carriers is used by the cell for production of all vital molecules such as amino acids, nucleobases, sugars and lipids. Polyphosphate chains directly regulate some processes in the cell and are used as phosphate donors in gene regulation. These two processes, energetic metabolism and regulation, are orchestrated by polyphosphate kinases. Polyphosphate kinases (PPKs) can currently be categorized into three groups (PPK1, PPK2 and PPK3) according their functionality; they can also be divided into three groups according their homology (EcPPK1, PaPPK2 and ScVTC). This review discusses historical information, similarities and differences, biochemical characteristics, roles in stress response regulation and possible applications in the biotechnology industry of these enzymes. At the end of the review, a hypothesis is discussed in view of synthetic biology applications that states polyphosphate and calcium-rich organelles have endosymbiotic origins from ancient protocells that metabolized polyphosphate.

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Specific localization of inorganic polyphosphate (poly P) in fungal cell walls by selective extraction and immunohistochemistry

Cited by 49 publications

References 39 publications

Polyphosphate is a cofactor for the activation of factor XI by thrombin

Polyphosphate is a cofactor for the activation of factor XI by thrombin

FTIR Spectroscopy for Evaluation and Monitoring of Lipid Extraction Efficiency for Oleaginous Fungi

Polyphosphate - an ancient energy source and active metabolic regulator

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