The pAb BL154G inhibits VWF-induced conformational changes in ADAMTS13.

<p>(A) ADAMTS13 was incubated with increasing concentrations of VWF for 30 min at 37°C followed by the addition of the pAb BL154G. (B) ADAMTS13 was incubated with BL154G antibody for 30 min at 37°C. Increasing concentrations of VWF were then added to the samples. Both sets of samples were analyzed by flow cytometry. Histograms represent binding of mAb in the presence of 0 (red), 100 (purple), 250 (green) and 500 (blue) µl of VWF stock. The figures represent data from one of three independent experiments. (C) Activity of ADAMTS13 in the absence (•) and presence (▪) of the pAb, BL154G, measured using the FRETS-VWF73 assay.</p

Flow cytometry based assay to detect intracellular ADAMTS13.

<p>(A) HEK293 cells transiently transfected with ADAMTS13 plasmid DNA were permeabilized and incubated with the anti ADAMTS13 antibodies Wh2-22-1A or Wh2-11-1 and an isotypic control antibody Anti-mouse IgG2a. The cells were washed to remove excess antibody and treated with an Alexa Flour 488 labeled secondary antibody. The fluorescence associated with the Alexa Flour 488 was measured in a flow cytometer and histograms showing the distribution of fluorescence intensity in a population of 10,000 cells following the different treatments are depicted. Cells treated with the control antibody show low levels of fluorescence that establishes the baseline for nonspecific interactions with the control and secondary antibodies. Histograms show permeabilized HEK293 cells incubated with the isotypic control antibody (red line), Wh2-22-1A (blue line) or Wh2-11-1 (green line) prior to incubation with the secondary antibody. (B) Apparent affinity and reactivity of the anti-ADAMTS13 mAbs Wh2-22-1A (•) and Wh2-11-1 (▪) was estimated using the flow cytometry based assay depicted in (A) above. The cells were incubated with increasing amounts of the antibodies and the median fluorescence (of histograms similar to those depicted in (A) above) were plotted as a function of µg antibody.</p

The activity is secreted and intracellular ADAMTS13.

<p>Activity of secreted (•) and intracellular ADAMTS13 (▪). Activity of intracellular ADAMTS13 was measured using the FRETS-VWF73 assay described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006506#pone-0006506-g004" target="_blank">Fig. 4</a>. The secreted ADAMTS13 was collected from the media which was concentrated using a Centriprep 30 centrifrugal filter device. The data were corrected for amount of ADAMTS13 in each sample as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006506#pone-0006506-g004" target="_blank">Fig. 4</a>. The activities for the secreted and intracellular ADAMTS13 were 17.3 arbitrary units/min/unit protein and 8.8 arbitrary units/min/unit protein respectively. Typical traces from three independent experiments are depicted.</p

VWF increases the immunoreactivity of anti-ADAMTS13 antibody Wh2-11-1.

<p>(A) Serial dilutions of the mAb Wh2-11-1 were allowed to bind to ADAMTS13 from human embryonic cells (HEK293 cells) in the absence (▪) or presence (•) of 100-500 µl from VWF stock. (B) Either 1 µg (▪) or 5 µg (•) of the mAb Wh2-11-1 were allowed to bind ADAMTS13 in the presence of increasing concentrations of VWF. The binding of the antibody in both experiments was monitored using flow cytometry and the data plotted using the curve-fitting software Graphpad Prism. (C) The binding of the mAb Wh2-11-1 was compared to that of the anti-V5 antibody in the absence or presence of VWF using flow cytometry. The histograms represent: Wh2-11-1 alone (red), Wh2-11-1 in the presence of VWF (green), anti-V5 alone (pink) and anti-V5 in the presence of VWF (blue). The figures represent data from one of three independent experiments.</p

VWF induces conformational changes in ADAMTS13 at 37°C which are inhibited by prior labeling with the mAb Wh2-11-1.

<p>ADAMTS13 from HEK293 cells was incubated with the mAb Wh2-11-1 and VWF under the following conditions: (I) ADAMTS13 was treated with VWF at 37°C prior to addition of Wh2-11-1. (II) ADAMTS13 was treated with Wh2-11-1 at 37°C followed by the addition of VWF. (III) ADAMTS13 was treated with VWF at 4°C prior to addition of Wh2-11-1. (IV) ADAMTS13 was treated with Wh2-11-1 at 4°C followed by the addition of VWF. *Reactivity of ADAMTS13 to Wh2-11-1 is significantly higher (p<.001) in the presence of VWF at 37°C. **There is no significant difference between the reactivity of Wh2-11-1 to ADAMTS13 in the presence or absence of VWF at 4°C (p = 0.083). Binding of mAb was monitored by flow cytometry. The figure represents data from one of five independent experiments.</p

A functionally deficient mutant of ADAMTS13 shows minimal conformation change in the presence of VWF.

<p>(A) Activity of wild-type (▪) and the P475S mutant (•) ADAMTS13s were measured by the FRET-VWF73 assay as described previously. Inset shows the activity of wild-type (filled bar) and the mutant (empty bar) ADAMTS13. (B) Wild-type and the P475S mutant ADAMTS13 were incubated with either BL154G alone (filled bars) or in the presence of 500 µl of VWF stock (empty bars). The VWF was incubated with ADAMTS13 prior to addition of the polyclonal Ab. Binding of polyclonal Ab was monitored by flow cytometry. The figures represent data from one of three independent experiments.</p

Heme Binding by <i>Corynebacterium diphtheriae</i> HmuT: Function and Heme Environment

The heme uptake pathway (hmu) of <i>Corynebacterium diphtheriae</i> utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV–visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT–CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A–CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT

Conservation of ADAMTS13 variants across species.

*<p>Synonymous ADAMTS13 variants.</p><p>Conservation scores were calculated using tools from the ConSeq server (<a href="http://conseq.tau.ac.il/ver1.1/index.html" target="_blank">http://conseq.tau.ac.il/ver1.1/index.html</a>). The conservation ranges from 1 (not conserved) to 9 (most conserved).</p

Specific activity of ADAMTS13 variants and limited proteolysis with trypsin.

<p><b>A.</b> Specific activity of twelve ADAMTS13 variants vs. WT as determined using FRETS-VWF73 substrate: Samples containing equal amounts of total extracellular protein were incubated with 5 µM of FRETS-VWF73 substrate and fluorescence readings were taken at 5 min intervals over the course of 2 h. Specific activity was calculated as the increase in relative fluorescence units (RFU)/min per unit of ADAMTS13 in the total extracellular protein samples. Extracellular ADAMTS13 protein amounts were determined using immunoblot analysis with anti-V5 antibody as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038864#pone-0038864-g002" target="_blank">Figure 2</a>. ADAMTS13 variants harboring synonymous variants are marked with (*) (**p-value <0.05). <b>B.</b> Expression and Michaelis-Menten Kinetics of non-synonymous variant 2699 vs. WT protein: HEK293 cells were transfected with WT and non-synonymous variant 2699 and harvest 24 h. post-transfection. Samples containing equal total protein amounts, both intracellular (I) and extracellular (E) (40 µg each), were separated by SDS-PAGE and probed with anti-V5 antibody to determine ADAMTS13 expression. Intracellular (•) and extracellular (▴) protein samples were incubated with 0–10 µM FRETS-VWF73 substrate. Fluorescence released upon cleavage was plotted over time, as previously described, and a best-fit linear regression was calculated to determine the initial rate of the reaction. The change in RFU per unit time was then used to estimate the specific activity of ADAMTS13. Specific activity at each substrate concentration was plotted and a Michaelis-Menten plot was generated using GraphPad Prism software to calculate K_M and V_max values. <b>C.</b> Limited Trypsin digestion of ADAMTS13 variants: Resistance of the intracellular full-length ADAMTS13 non-synonymous variant 1852 (▴) was compared to that of WT (▪) using densitometry analysis of the immunoblot data, as described the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038864#s4" target="_blank">Materials and Methods</a></i>. Non-synonymous variant 1852 revealed increase resistance to trypsin digestion compared to WT.</p

ADAMTS13 synonymous and non-synonymous variants investigated in this study.

*<p>Synonymous ADAMTS13 variants.</p>**<p>Based on SwissProt annotation of domains and regions.</p