Journal: PLoS ONE

Article Title: Proteomic Analysis of Mitochondrial-Associated ER Membranes (MAM) during RNA Virus Infection Reveals Dynamic Changes in Protein and Organelle Trafficking

doi: 10.1371/journal.pone.0117963

Figure Lengend Snippet: (A) Venn diagram of proteins that share a MAM-localization profile similar to that of MAVS. Proteins that move out of the MAM fraction during HCV (greater than 2-fold change over mock with a Benjamini-Hochberg corrected p-value of < 0.05) were compared to those that either move into the MAM or remain localized to the MAM during SenV infection. The 281 proteins in the intersection share this MAVS-localization pattern in the MAM during RNA virus infection. (B) Heat map of the 281 proteins with “MAVS-like” localization identified in MAM fractions from HCV and SenV, as well as mock (see panel A). Log 10 -transformed spectral counts across technical triplicates are represented by the color intensity shown in the key. (C) Immunoblot analysis of lysate (Input) and anti-Myc immunoprecipated extracts (Pellet) from cells expressing Myc-MAVS (+) or vector (-), and either Flag-RAB1B, Flag-VTN, or Flag-LONP1. (D) IFN-β promoter reporter luciferase expression of Huh7 cells expressing empty vector or Flag-LONP1 and then mock or SenV infected (24h). Values are mean -/+ SD (n = 3) of one of three replicate experiments; **P<0.01. (Inset) Immunoblot for Flag-LONP1 and tubulin (Tub.) protein expression.

Article Snippet: Plasmids encoding human VTN (NM_000638.3), LONP1 (NM_004793.2), and RAB1B (NM_030981.1) were obtained (Origene).

Techniques: Infection, Transformation Assay, Western Blot, Expressing, Plasmid Preparation, Luciferase

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: A transient ternary “Molecular Sandwich” type complex (MSC) is a part of the PAI-1 inhibitory mechanism in vivo.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: In Vivo

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Crystal structures of PAI-1 (yellow) complexes with S195A tcuPA (blue) (33), and SMB domain of Vn (brown) (34) were used. The exposed RCL of active PAI-1 is shown in red with positions of E350 and E351 (P4′P5′ nomenclature of Schechter and Berger (53)) in blue, β-sheet A in green and a-helix F in cyan. Active site A195 of S195A tcuPA is shown as a white space-filled residue, and positions of positively charged residues of 37-loop of uPA are red.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Scheme 3: A transition intermediate (I‡), which is in equilibrium with the active PAI-1 (IA), forms during conformational changes accompanying the transition to the latent conformation (IL) (46). A diagram depicts how IA is stabilized in the complex with ligand(s) (IA1) due to an increase in the ΔG‡ (ΔΔG‡ = ΔG‡1 - ΔG‡0), where ΔG‡1 and ΔG‡0 are changes in the activation Gibbs free energy for active PAI-1 with and without ligand(s), respectively. A bar graph shows the contribution of Vn (black), S195A tcuPA (white), and both ligands (gray) to ΔΔG‡ for rPAI-1, Q123K PAI-1, and Gl-PAI-1 at 37°C. The values of ΔG‡0 and ΔG‡1 were calculated as ΔG‡ = ΔH‡ - TΔS‡ (Table 3).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Activation Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Linear dependences of kobs from [ligand] for the association of PAI-1 and its complexes with ligands (k+n; n=1–4; Scheme 1). Values of k+n for the interaction of S195A tcuPA with NBD P1′ PAI-1 (○); Vn and NBD S119C PAI-1 (□); S195A tcuPA with NBD P1′ PAI-1/Vn (●), and Vn with NBD S119C PAI-1/S195A tcuPA (■) were calculated from the slopes of the best linear fit to the data (Table 1). Slow dissociation of S195A tcuPA limits the reaction between tPA and complex of NBD P9 PAI-1 with S195A tcuPA (△) and MSC (complex of NBD P9 PAI-1 with S195A tcuPA and Vn; Scheme 1) (▲).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The values of the association and dissociation rate constants (k +1 , k +2 , k +3 , k +4 and k −1 , k −2 , k −3 , k −4 respectively); and dissociation equilibrium constants (K D = k − /k + ) for the interactions between wt PAI-1, its mutant variants, and their complexes with S195A tcuPA and Vn at 25°C.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Mutagenesis, Variant Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The dependences of kobs for the fast step (an increase in NBD fluorescence emission) (○), and slow step (a decrease in the NBD fluorescence emission) (●) on enzyme concentration. The solid lines represent the best fit of the linear equation kobs2 = (klim+k−1) + k1[tcuPA] (r2=0.93) (fast step), and a hyperbolic equation kobs1 = klim*[tcuPA]/(Km + [tcuPA]) (r2=0.99) (slow step) to the data by SigmaPlot 11.0. The values of klim and Km (Scheme 2) were 33.3 ± 5.2 s−1, and 10.2 ± 2.2 μM, respectively. The slope and intercept of the linear dependence of kobs on enzyme concentration represent a second order rate constant for the interaction of tcuPA with NBD P1′ PAI-1 k+1 = 26.8 ± 2.8 μM−1 s−1, and (klim+k−1) = 50 ± 8 s−1, respectively. Inset: Trace of the changes in NBD fluorescence for the reaction of NBD P1′ PAI-1 (0.12 μM) with 2.0 μM tcuPA. Values of kobs1 and kobs2 were calculated by fitting (grey line) a double exponential equation to the time traces of changes in NBD-fluorescence emission using SigmaPlot 11.0.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Fluorescence, Concentration Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Two step inactivation of proteianase (E) by PAI-1 (I)

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: (A) SDS PAGE analysis of products of the reaction between sctPA and rPAI-1, Q123K PAI-1, and Gl-PAI-1 and their complexes with Vn, S195A tcuPA, and both ligands (the table at the top of the gels.) incubated for 0, 1, 15, 90 (stained with SYRRO Ruby) and 720 h (PAI-1 antigen was visualized with Western blot analysis) at 37°C. Positions of PAI-1/tPA SIC (1), Vn (2), tPA (3), S195A tcuPA (4), latent and cleaved rPAI-1 (5 and 6, respectively), co-migrating S195A tcuPA and latent Gl-PAI-1 (7), cleaved Gl-PAI-1 (8), are indicated to the left (rPAI-1 and Q123K PAI-1) and to the right (Gl-PAI-1) of the gels. (B) Active PAI-1 concentration in the reaction mixtures, shown in the Western blot (A, lower panel) was estimated by inhibiting the activation of Plg by uPA, as described elsewhere (9;41). After incubation with both Vn and S195A tcuPA for 1 month (720h) at 37°C rPAI-1 (filled bars) and Gl-PAI-1 (gray bars), but not Q123K PAI-1 (empty bars) inhibit the activation of Plg by uPA.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: SDS Page, Incubation, Staining, Western Blot, Concentration Assay, Activation Assay

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: The concentration of active PAI-1 was determined from the residual tPA amidolytic activity after titrating the sample with increasing amounts of sctPA as described under Experimental Procedures and plotted on a semilogarithmic scale (filled symbols). Separate aliquots were incubated with an excess of sctPA and the products of the reaction were separated by SDS PAGE, as shown in Figure 4 and described under Experimental Procedures. The amounts of active PAI-1 were estimated from relative density of SIC, latent and cleaved PAI-1 on gels and plotted as empty symbols. Linear equations were fit to both sets of data (solid lines represent the best fits to filled symbols) using SigmaPlot 11.0, and the first order rate constants (kL1, kL2, kL3, kL4 (Table 2), and kL1SDS PAGE, kL2SDS PAGE, kL3SDS PAGE, kL4SDS PAGE respectively, Scheme 1) were calculated from the slopes. Inset: Correlation between values of kL measured by two different methods. The values of kL estimated from the results of SDS PAGE analysis were plotted against the corresponding values obtained from titrating the reaction mixtures with sctPA: rPAI-1 (empty symbols), Gl-PAI-1 (black symbols), and Q123K PAI-1 (grey symbols). Data for free rPAI-1, Gl-PAI-1, and Q123K PAI-1 are shown as circles, for their complexes with Vn as triangles, with S195A tcuPA as reversed triangles, and for both ligands as squares. The solid line represents the best linear fit to the data of linear equation (kLSDS PAGE=−0.27+ 0.93 kL; r2= 0.99).

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Concentration Assay, Activity Assay, Incubation, SDS Page

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: Values of k L1 , k L2 , k L3 and k L4 ( Scheme 1 ) for rPAI-1, Q123K PAI-1, Gl-PAI-1 and their complexes with Vn, S195A tcuPA and both ligands, determined at different temperatures.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: A linear correlation between changes in the contribution of -TΔS‡ and ΔH‡ (Table 3) to ΔG‡ for the three PAI-1 variants and their complexes with ligands at 37°C. The values of -TΔS‡ for rPAI-1 (empty symbols), Gl-PAI-1 (black symbols), and Q123K PAI-1 (grey symbols) for free serpins (circles), and their complexes with Vn (triangles), S195A tcuPA (reversed triangles), and both ligands (squares) were plotted as a function of ΔH‡. The solid line represents the best fit of the linear equation (-TΔS‡ =90.6-0.94 ΔH‡; r2=0.99) to the data.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques:

Journal: Biochemistry

Article Title: Remarkable Stabilization of Plasminogen Activator Inhibitor 1 in a "Molecular Sandwich" Complex

doi: 10.1021/bi400470s

Figure Lengend Snippet: MSCs formed with S195A tcuPA and mAbs protect rPAI-1/Vn from inactivation by MA-33B8 and spontaneous active to latent transition. Dependences of kobs for RCL insertion for NBD P9 PAI-1 (○), NBD P9 PAI-1/Vn (●) and MSCs with MA-56A7C10 (△), MA-44E4 (□), and S195A tcuPA (▽) on [MA-33B8]. NBD P9 PAI-1, its complex with Vn (10 nM), and MSCs formed in presence of 20 nM of S195A tcuPA or a mAb (MA-56A7C10 or MA-44E4) were incubated with MA-33B8 (50–400 nN) in 96-well flat bottom plates from Costar (Corning Inc) and an increase in NBD fluorescence with time was registered using a fluorescence spectrophotometer SynergyTM HT Hybrid Reader. The kobs were calculated by fitting a single exponential equation to the data using Gen5™ 2.0 Data Analysis Software (BioTek) and SigmaPlot 11.0 (SPSS Inc.), as described elsewhere (26;43;45). Inset: Stabilization of rPAI-1/Vn (lane A) in MSCs with S195A tcuPA (lane B), MA-56A7C10 (lane C), and MA-42A2F6 (lane D) after incubation at 37°C for 168 h (one week). Active PAI-1 was quenched by sctPA and reaction mixtures were analyzed as described under Experimental Procedures. Positions of mAb (1), PAI-1/tPA SIC (2), Vn (3), tPA (4), S195A tcuPA (5), latent (6) and cleaved PAI-1 (7) are indicated to the right of the gel.

Article Snippet: Monomeric Vn, wt non-glycosylated (r), glycosylated (Gl-) PAI-1, non- glycosylated Q123K PAI-1 (lacks vitronectin binding), and three mutant variants of PAI-1 with introduced cysteines labeled with N-((2-(iodoacetoxy) ethyl)-N-methyl) amino-7-nitrobenz-2-oxa-3-diazole (NBD) - S338C (NBD P9) PAI-1, M447C (NBD P1′) PAI-1 and S119C (NBD S119C) PAI-1 were purchased from Molecular Innovations (Novi, MI).

Techniques: Incubation, Fluorescence, Spectrophotometry, Software

Journal: Cell Reports Methods

Article Title: A stress-reduced passaging technique improves the viability of human pluripotent cells

doi: 10.1016/j.crmeth.2021.100155

Figure Lengend Snippet:

Article Snippet: We also cultured human PSCs on recombinant human vitronectin (20-388 a.a.) (rhVTN, WAKO) in TeSR-E8 media (Veritas) ( ; ).

Techniques: Recombinant, Knock-Out, Lysis, TaqMan Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Ligation, Mass Spectrometry, Software, Cell Counting

Journal: Scientific Reports

Article Title: Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5

doi: 10.1038/s41598-018-23194-9

Figure Lengend Snippet: Phagocytosis assay by THP-1 cells. ( a ) (i) Western blotting of bound protein(s) on latex beads after solubilization. Lane 1: SE36-beads, lane 2: SE36-beads with VTN, lane 3: latex-beads alone, lane 4: beads with VTN. SE36 and VTN bound to the beads were detected by anti-SE36 mouse serum (diluted 1:1000) and anti-VTN pAb (diluted 1:2000), respectively. Arrows indicate the target proteins. (ii) Representative flow cytometry (FCM) histogram of the antibody-independent engulfment of SE36-beads with or without VTN by THP-1 cells. (iii) Giemsa staining of engulfed SE36-beads (left panel) and non-treated beads (right panel) by THP-1 cells. Arrows indicate the engulfed SE36-beads. Scale bar, 10 μm. ( b ) FCM histogram of the engulfment by THP-1 cells of SE36-beads with or without VTN and/or purified IgG from NHS or HTS. ( c ) FCM histogram of the engulfment by THP-1 cells of SE36-beads treated with normal serum or VTN-depleted serum (VTN-depl). ( d ) FCM histogram of the engulfment by THP-1 cells of SE36-beads with or without VTN-depl, with or without purified VTN or with normal serum.

Article Snippet: Purified VTN was purchased from Promega (Madison, WI, USA), and VTN recombinants, Recombinant Human Vitronectin (62–398 aa) Protein (Cat #: HRP-0323) and Recombinant Human Vitronectin Protein, CF (Cat #: 2308-VN), were obtained from LD Biopharma (San Diego, CA, USA) and R&D systems (Boston, MA, USA), respectively.

Techniques: Phagocytosis Assay, Western Blot, Flow Cytometry, Staining, Purification

Journal: Scientific Reports

Article Title: Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5

doi: 10.1038/s41598-018-23194-9

Figure Lengend Snippet: Binding assay of vitronectin (VTN) to SE36. ( a ) Reactivity of the purified VTN and HSA against SE36. SE36 was adsorbed to microtiter plate and VTN or human serum albumin (HSA) was added at the indicated concentrations. ( b ) Western blotting of elutes from SE36-immobilized and control columns. The anti-VTN pAb detected two forms (V75 and V65; upper band and lower band, respectively) of VTN. Each elute was run in an SDS-PAGE gel and probed with anti-VTN pAb (diluted 1:2000). ( c ) Reactivity of VTN in naïve human serum (NHS) and Ugandan high anti-SE36 IgG-titer serum (HTS) against SE36. SE36 was adsorbed to microtiter plate and NHS or HTS was added at the indicated dilutions (closed symbols). Open symbols show reactions without anti-VTN Ab as negative control. ( d ) Reactivity of commercially available VTN recombinants (see also Fig. 1e) against SE36. SE36 was adsorbed to microtiter plate and VTN recombinants were added at the indicated concentrations. ( e ) Schematic representation of VTN. “SmB”, “Hp”, and “Hb” indicate Somatomedin-B motif, Hemopexin domain, and Heparin-binding region, respectively. The number 398 denotes the endogenous cleavage site. The designations “20–478” and “62–398” indicate commercially available VTN recombinants. VTN-1 to -4, as well as VTN-2-1 to -2-3 denote truncated recombinant forms. Results in ( a , c , and d ) are expressed as means ± SD from three independent experiments. In ( c ), statistical analysis between NHS and HTS was performed using a Mann-Whitney U test. No significant differences were found.

Article Snippet: Purified VTN was purchased from Promega (Madison, WI, USA), and VTN recombinants, Recombinant Human Vitronectin (62–398 aa) Protein (Cat #: HRP-0323) and Recombinant Human Vitronectin Protein, CF (Cat #: 2308-VN), were obtained from LD Biopharma (San Diego, CA, USA) and R&D systems (Boston, MA, USA), respectively.

Techniques: Binding Assay, Purification, Western Blot, Control, SDS Page, Negative Control, Recombinant, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5

doi: 10.1038/s41598-018-23194-9

Figure Lengend Snippet: Mapping of binding site of VTN in SE36. ( a ) Schematic representation of SE36. The “8” and “S” indicate octamer repeat and serine rich region, respectively. “RGD” indicates RGD motif. The number 178 denotes the position of polyserine sequence present in PfSERA5 but deleted in SE36 . SE36-1 to -4 denote truncated recombinant forms. P1 to P15 are synthetic peptides . ( b ) Reactivity of VTN in NHS and HTS against each truncated SE36 recombinant. Each recombinant was adsorbed to microtiter plate and NHS and HTS were added at 1:2000 dilution. ( c ) Reactivity of the purified VTN against each truncated SE36 recombinant. Each recombinant was adsorbed to microtiter plate and the purified VTN was added at 2 μg/mL concentration. ( d ) Reactivity of VTN in NHS and HTS against each synthetic peptide. Each synthetic peptide was adsorbed to microtiter plate (0.3 μM) and NHS and HTS were added at 1:2000 dilution. ( e ) Reactivity of the purified VTN against each synthetic peptide. Each synthetic peptide was adsorbed to microtiter plate and the purified VTN was added at 2 μg/mL concentration. Results in ( b – e ) are expressed as means ± standard deviation (SD) from three independent experiments. In ( b and d ), statistical analyses between NHS and HTS were performed using a Mann-Whitney U test. No significant differences were found.

Article Snippet: Purified VTN was purchased from Promega (Madison, WI, USA), and VTN recombinants, Recombinant Human Vitronectin (62–398 aa) Protein (Cat #: HRP-0323) and Recombinant Human Vitronectin Protein, CF (Cat #: 2308-VN), were obtained from LD Biopharma (San Diego, CA, USA) and R&D systems (Boston, MA, USA), respectively.

Techniques: Binding Assay, Sequencing, Recombinant, Purification, Concentration Assay, Standard Deviation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Molecular Camouflage of Plasmodium falciparum Merozoites by Binding of Host Vitronectin to P47 Fragment of SERA5

doi: 10.1038/s41598-018-23194-9

Figure Lengend Snippet: Characterization of VTN binding to SE36. ( a ) Biophysical interaction analysis of the binding of VTN to SE36 using surface plasmon resonance (SPR). The raw data (color lines) were fitted to 1:1 binding and bivalent analyte model (black lines) using the Biacore T200 Evaluation software. The fit parameters from these models are summarized in ( b ). These experiments were performed in triplicate, and representative data are shown. ( b ) Kinetic and equilibrium constants for the binding of VTN to SE36. ( c ) Schematic representation of two schemes in bivalent binding model. (i) VTN contains two binding sites. (ii) VTN contains a single binding site and forms a dimer. ( d ) (i) Purity of VTN-1 to -4 recombinants confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. Two micrograms of each recombinant was run in a gel. Arrows indicate target proteins. Since estimated molecular weights were 18, 21, 14, and 12 kDa in VTN-1 to -4, respectively, it was presumed that these recombinants were glycosylated in Pichia pastoris . (ii) Reactivity of SE36 against each truncated VTN recombinant. Each recombinant was adsorbed to microtiter plate (1 μg/mL) and SE36 was added at the indicated concentrations. ( e ) (i) Purity of VTN-2-1 to -2-3 recombinants confirmed by SDS-PAGE and CBB staining. Two micrograms of each of recombinant was run in a gel. Arrows indicate target proteins. Since estimated molecular weights were 8 kDa in VTN-2-1 to -2-3, it was, likewise, presumed that these recombinants were glycosylated in P. pastoris . All recombinant proteins of VTN ( d and e ) were detected by anti-His tag antibody in western blotting and was purified by His GraviTrap. (ii) Reactivity of SE36 against each truncated VTN recombinant. Each recombinant was adsorbed to microtiter plate (1 μg/mL) and SE36 was added at the indicated concentrations.

Article Snippet: Purified VTN was purchased from Promega (Madison, WI, USA), and VTN recombinants, Recombinant Human Vitronectin (62–398 aa) Protein (Cat #: HRP-0323) and Recombinant Human Vitronectin Protein, CF (Cat #: 2308-VN), were obtained from LD Biopharma (San Diego, CA, USA) and R&D systems (Boston, MA, USA), respectively.

Techniques: Binding Assay, SPR Assay, Software, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Recombinant, Western Blot, Purification