Journal: Kidney360

Article Title: Removal of Middle Molecules and Dialytic Albumin Loss: A Cross-over Study of Medium Cutoff and High-Flux Membranes with Hemodialysis and Hemodiafiltration

doi: 10.34067/KID.0000000000000185

Figure Lengend Snippet: Reduction ratios (in percentage of initial concentration) during the treatments in the four study phases. Study phases: HFX-HD, high-flux hemodialysis; HFX-HDF, high-flux hemodiafiltration; MCO-HD, hemodialysis using a medium cutoff dialyzer membrane; MCO-HDF, hemodiafiltration using a medium cutoff dialyzer membrane.

Article Snippet: In other words, the results in our study demonstrate that a similar performance of Theranova 400 (surface area 1.7 m 2 ) on B2M removal can be achieved when compared with a HFX dialyzer with a larger membrane surface area (2.5 m 2 ) without a larger pore size and with a significantly higher albumin loss.

Techniques: Concentration Assay, Membrane

Journal: Kidney360

Article Title: Removal of Middle Molecules and Dialytic Albumin Loss: A Cross-over Study of Medium Cutoff and High-Flux Membranes with Hemodialysis and Hemodiafiltration

doi: 10.34067/KID.0000000000000185

Figure Lengend Snippet: Cumulative albumin and total protein loss during treatments for four study phases. Study phases: HFX-HD, high-flux hemodialysis; HFX-HDF, high-flux hemodiafiltration; MCO-HD, hemodialysis using a medium cutoff dialyzer membrane; MCO-HDF, hemodiafiltration using a medium cutoff dialyzer membrane.

Article Snippet: In other words, the results in our study demonstrate that a similar performance of Theranova 400 (surface area 1.7 m 2 ) on B2M removal can be achieved when compared with a HFX dialyzer with a larger membrane surface area (2.5 m 2 ) without a larger pore size and with a significantly higher albumin loss.

Techniques: Membrane

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: EGF1 of human coagulation factor X undergoes AspH-catalysed hydroxylation at Asp103 hFX . MS/MS-analysis was performed using a Bruker Daltonics amaZon TM Ion Trap LC-MS/MS system equipped with a Dionex TM UltiMate ® 3000 HPLC machine. Endpoint turnover assays were performed under standard (non-redox) conditions (for details see Methods section). For MS/MS-analysis, after quenching, cystine disulfides were reduced (dithiothreitol) and cysteine thiols derivatized with iodoacetamide. a Reaction scheme for diastereospecific AspH-catalysed hydroxylation of Asp/Asn-residues in EGFDs. b Schematic structure and calculated mass of the expected disulfide isomer of hFX EGF1 39mer (aa 86–124) featuring a canonical disulfide connectivity pattern (Cys1–3, 2–4, 5–6; green); the hydroxylation site (Asp103 hFX ) is in red. c His 6 -AspH 315–758 catalysed hydroxylation of hFX EGF1 39mer proceeds with ~40% conversion under standard (non-redox) conditions; the light orange graph represents a control in which AspH was replaced by buffer. d LC-MS/MS analysis of the relevant digestion fragment of hFX EGF1 39mer (aa 101–119), which is partially hydroxylated (~40%) after treatment with His 6 -AspH 315–758 , confirms AspH-catalysed hydroxylation takes place at Asp103 hFX as anticipated based on the AspH-substrate consensus sequence (fragments bearing β-hydroxy-Asp are in red). e LC-MS/MS analysis of the relevant digestion fragment of hFX EGF1 39mer (aa 101–119) before exposure to His 6 -AspH 315–758

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Coagulation, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Control, Sequencing

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: Structure of AspH-TPR-Ox:hFX features a non-canonical EGFD disulfide connectivity. Color code: magenta: carbon-backbone of NC 39mer peptide; yellow: carbon-backbone of N -oxalylglycine (NOG); violet: Mn; red: oxygen; blue: nitrogen; pale yellow: sulfur. a Overview of the AspH-TPR-Ox:hFX structure; His 6 -AspH 315–758 in grey. b Surface representation (grey) of the AspH-TPR-Ox:hFX structure showing the substrate-binding groove, His 6 -AspH 315–758 in green. c Interactions of the TPR domain residues His493 and Arg526 as well as the hinge region residue Tyr565 with the NC 39mer peptide. d Additional interactions of the TPR domain with the NC 39mer peptide: Asn395 forms a hydrogen bond with Phe116 hFX (3.0 Å) and Phe432 forms a hydrogen bond with Gly115 hFX (3.2 Å). e OMIT electron density map (mFo–DFc) contoured to 3σ around the hFX derived NC 39mer peptide supports the presence of a non-canonical disulfide bridge between Cys101 hFX and Cys110 hFX as a substrate requirement for AspH (canonical EGFD disulfide isomer: Cys1–3, 2–4, 5–6; non-canonical EGFD disulfide isomer: Cys1–2, 3–4, 5–6; see Fig. ). Note that electron density for two alternative conformation of the Asp103 hFX side chain is observed

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Binding Assay, Residue, Derivative Assay

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: Significant conformational changes occur in AspH upon substrate binding. Color code: grey: His 6 -AspH 315–758 (AspH-TPR-Ox:hFX); turquoise: His 6 -AspH 315–758 (AspH-TPR-Ox); magenta: carbon-backbone of NC 39mer peptide; yellow: carbon-backbone of N -oxalylglycine (NOG); violet: Mn; red: oxygen; blue: nitrogen; pale yellow: sulfur. w: water. a Superimposition of the AspH-TPR-Ox:hFX (grey) and AspH-TPR-Ox (turquoise) structures indicate conformational changes in the TPR domain, the hinge region, and the oxygenase acidic loop on substrate binding. b Superimposition of the AspH-active sites of the AspH-TPR-Ox:hFX (grey) and AspH-TPR-Ox (turquoise) structures: The interaction between Glu617 apo and Arg620 (2.5 Å) in the AspH-TPR-Ox structure is lost on substrate binding; both Asp616 and Glu617 interact with the substrate in the AspH-TPR-Ox:hFX structure. Gln627 (3.2 Å) and Lys666 (2.7 Å) bind to the Asp103 hFX carboxylate of the active conformer of the AspH-substrate. On substrate binding, the side chain of Glu615 rotates by ~90° to interact with the side chain of Arg620 (2.8 Å) and the main chain of Leu619 (2.8 Å), rather than Lys666 (2.7 Å) as in the substrate unbound state. c The side chain of the Asp103 hFX residue undergoing hydroxylation is observed in two conformations (A: magenta and B: pink; see Supplementary Fig. for details). The Asp103 hFX side chain carboxylate of conformation B (pink) is positioned (2.6 Å) to interact with the Mn. d Close-up of the AspH-active site: The pro-R hydrogen at the Asp103 hFX β-position of the likely productive NC 39mer conformation A (magenta) is positioned to interact with the Mn (distance Cβ-Mn: 4.2 Å), consistent with hydroxylation at this position

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Binding Assay, Residue

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: AspH fully hydroxylates a mixture of canonical and non-canonical EGFD disulfides under redox conditions. End-point turnover reactions were performed as in the Methods Section. a Schematic structures of the two major hFX EGF1 39mer disulfide isomers identified in a single batch of hFX EGF1 39mer obtained by thiol oxidation in air-saturated buffer (see Supplementary Information); disulfides are in green (canonical isomer, Cys1–3, 2–4, 5–6, top; non-canonical isomer, Cys1–2, 3–4, 5–6, bottom), the hydroxylation sites (Asp103 hFX ) are in red. b >95% Hydroxylation of hFX EGF1 39mer was observed under redox conditions as opposed to partial hydroxylation under standard (non-redox) conditions (Fig. ), indicating that a ‘non-canonical’ EGFD-disulfide pattern (Cys 1–2, 3–4, 5–6) is the actual AspH-substrate; the light orange graph represents a control in which AspH was replaced by buffer

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Control

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: A cyclic peptide mimicking the non-canonical Cys3–4 EGFD is an excellent AspH-substrate. End-point turnover reactions were performed as in the Methods Section. a Schematic structure and calculated mass of the cyclic peptide CP 101–119 mimicking the central macrocyclic disulfide of the non-canonical isomer of hFX EGF1 39mer (Cys3–4); the hydroxylation site (Asp103 hfX ) is in red. b >95% Hydroxylation of the CP 101–119 peptide was observed under standard (non-redox) conditions; the light orange graph represents a control in which AspH was replaced by buffer. c Two His 6 -AspH 315–758 -molecules are present in the asymmetric unit of the AspH-TPR-Ox:CP 101–119 crystal structure, only one binds the CP 101–119 peptide (details in the Supplementary Information): The superimposition of the two independent AspH-molecules of this crystal structure (colour code: unbound AspH: raspberry; AspH bound to CP 101–119 : grey; CP 101–119 : green) highlights an induced fit mechanism of AspH upon substrate binding: Major conformational changes occur in the TPR domain, the hinge region and the acidic loop whilst the overall oxygenase domain conformation is hardly affected. The thioether linker of the cyclic peptide could not be accurately modelled into the electron density; however, complementary analytical methods imply the presence of a cyclic thioether linkage (see Supplementary Figure 16 for further details)

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Control, Binding Assay

Journal: Nature Communications

Article Title: Aspartate/asparagine-β-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern

doi: 10.1038/s41467-019-12711-7

Figure Lengend Snippet: The AspH-substrate requirement is a ring composed of 10 amino acid residues. End-point turnover reactions were performed as in the Methods Section. Light orange graphs represent controls in which AspH was replaced by buffer. a Schematic structure and calculated mass of the NC-4Ser 39mer peptide featuring a single disulfide between Cys3–4 (green); its sequence is identical to the one of hFX EGF1 39mer (see Fig. ), except that Cys90 hFX , 95, 112, 121 are substituted for Ser (light blue) to avoid disulfide scrambling; the hydroxylation site (Asp103 hFX ) is in red. b >95% Hydroxylation was observed under standard (non-redox) conditions. c >95% Hydroxylation was observed under redox conditions. d Superimposition of the AspH-TPR-Ox:hFX crystal structure (color code: AspH: grey; NC 39mer peptide: magenta) with the AspH-TPR-Ox:NC-Ser 39mer crystal structure (color code: AspH: gold; NC-4Ser 39mer peptide: slate blue) shows a high conservation of the conformations of both enzyme and ligands

Article Snippet: Derivatives of hFX EGF1 39mer containing either canonical (C 26mer ) or non-canonical (NC 26mer ; NC-4Ser 39mer ) disulfides were synthesized as single disulfide isomers by GL Biochem (Shanghai) Ltd using orthogonal cysteine protection.

Techniques: Sequencing