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

Ribbon diagrams of the ADAMTS13 fragments.

<p>Two upper panels: Left - comprising residues (77–470) and right (674–1254) built using comparative modeling (3D-PSSM algorithm). Bottom panel ADAMTS13 X-ray structure residues (287–682) PDB ID 3GHM. Helices are depicted in red and beta-structures are in yellow. Residues encoded by codons/variants under investigation are denoted with residue number (van der Waals’ radii of the side chains are shown). Beginning and end of each fragment are also denoted with a residue number.</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

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

Analysis of mRNA structure/stability, codon usage and expression levels of ADAMTS13 variants: A.

<p>mFold ΔΔG values of <i>ADAMTS13</i> mRNA fragments: RNA fragments of 25, 75, 151, and 399 nucleotides in length were queried using online mFold server, utilizing default server settings. Variants were centered within the RNA fragments used in the analysis. The most stable structures (lowest ΔG) for both variant and WT <i>ADAMTS13</i> variants were chosen. ΔΔG (ΔG variant - ΔG WT) was calculated and is displayed here for each of the variants. <b>B.</b> KineFold ΔΔG values calculated using online KineFold server (obtained employing a similar strategy). <b>C. </b><i>ADAMTS13</i> mRNA expression levels as determined by qPCR: Analysis of mRNA expression was performed 24 h. post-transfection with quantitative real time PCR on WT <i>ADAMTS13</i> and all twelve variants. <i>GAPDH</i> was used as a reference and a ΔΔCp was calculated using the average of all WT ΔCp results for comparison. Fold change relative to WT is presented on a logarithmic scale. mRNA expression levels were analyzed in multiple independent transfection experiments utilizing each variant and WT <i>ADAMTS13</i> variant, and found to be consistently similar from one experiment to the another (**p-value <0.05). <b>D.</b> Differences in Relative Synonymous Codon Usage (RSCU) values between each of the <i>ADAMTS13</i> variant codons and the WT codon: ΔRSCU =  Δ(RSCUVariant – RSCUWT); the more positive is the ΔRSCU value, the more common is the variant codon compared to WT codon. RSCU values were calculated using the ADAMTS13 coding region and human codon usage information. <b>E.</b> Differences between log ratio of codon usage values for each of the <i>ADAMTS13</i> variants and WT. The plotted values are the differences between log ratio of codon usage frequency of the variant and WT codon (Δ(variant – WT)). The more positive the delta, the more commonly used the variant codon is, compared to WT. Values were calculated using the ADAMTS13 coding region and human codon usage information. <b>F.</b> Normal distribution of RSCU values for all variants in <i>ADAMTS13</i>: The RSCU and log ratio of codon usage values were determined for all variants in the coding region of <i>ADAMTS13</i>. MAD scores were assigned to the ΔRSCU and Δlog ratio of all variants. Comparison of MAD scores of twelve variants and all variants in <i>ADAMTS13</i> revealed that ΔRSCU and Δlog ratio scores for the twelve variants fell within the distribution of variants in <i>ADAMTS13</i>. <i>ADAMTS13</i> variants harboring synonymous variants are marked with (*).</p