MOESM3 of Plasmodium vivax msp-3Îą polymorphisms: analysis in the Indian subcontinent

Additional file 3: Table S2 Plasmodium vivax Alu and HhaI MSP3a alleles and their frequencies in the Indian subcontinent

MOESM1 of Plasmodium vivax msp-3α polymorphisms: analysis in the Indian subcontinent

Additional file 1: Figure S1 1 (A) Polymerase chain reaction amplified fragments of Pvmsp-3α gene from P. vivax field isolates. Digestion pattern using Restriction fragment length polymorphism of Pvmsp-3α using AluI (B) and HhaI (C) restriction enzymes. 1kb and 100bp DNA markers

MOESM2 of Plasmodium vivax msp-3Îą polymorphisms: analysis in the Indian subcontinent

Additional file 2: Table S1 Pvmsp-3Îą RFLP pattern (Alu 1 and Hha1 restriction enzymes) and genotyping among P. vivax isolates collected from different cities in India

Alignment of falstatin with other ICPs.

<p>A multiple sequence alignment was performed with falstatin and ICPs from <i>Cryptosporidium parvum</i> (cryptostatin), <i>Trypanosoma cruzi</i> (chagasin), and <i>Plasmodium berghei</i> (PbICP). This alignment predicted four major loop regions; L0, BC (L2), DE (L4) and FG (L6) in falstatin. The peptides used in this study are underlines, and residues mutated in the described studies are in bold type.</p

Only the BC loop appears to plays a central role in protease inhibition.

<p>Peptides based on the DE, FG and L0 loops of falstatin also incubated with VP2 (Fig. 5A) or FP2 (Fig. 5B) or FP3 (Fig. 5C, Fig. 5D add E), and hemoglobin hydrolysis patterns were observed in 15% SDS-PAGE. As a control the wild type peptide was pre-incubated with trypsin, pepsin, and plasmepsin- 4 of <i>P.vivax</i> (PvPM4) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE (5F). The hydrolysis of hemoglobin and fluorogenic substrate was done with two independent batches of each enzyme.</p

Falstatin is a multimer.

<p>Native falstatin was characterized by gel filtration and native PAGE analysis. The elution profile of falstatin and its migration in native PAGE (right inset) predict a size of ∼450 kDa. Lane A: 660 kDa and 440 kDa markers; Lane B: 43 kDa marker; Lane C: 8 μg of active falstatin; Lane D: 12 μg of active falstatin. The functional activities of different peaks were analyzed by hemoglobin hydrolysis observed in 15% SDS-PAGE (left inset). Gel filtration analysis and activity assay of different peaks were repeated more than two times. The blue and red lines indicate the absorbance of proteins and conductance of buffer, respectively.</p

Effect of BC peptide on cultured <i>P. falciparum</i>. A Morphology of parasites treated with wild type and mutant BC peptides.

<p>Synchronized ring-stage parasites were incubated with the wild type (LDSVN²⁸⁷G<b>N²⁸⁹</b>G<b>F²⁹¹</b>VW) and the mutated peptide (LDSVN²⁸⁷G<b>A²⁸⁹</b>G<b>F²⁹¹</b>VW) (25.0 μM) or E-64 (10 μM), and after incubation parasites were stained with Giemsa. Morphology of parasites was analyzed with two independent cultures of <i>P. falciparum</i>. B. <b>Impact of wild type BC peptide on free heme formation.</b> Synchronized ring-stage parasites were incubated without inhibitor or with the peptide or E-64 at the indicated concentrations, and after 30 hours free heme was quantified by spectroscopy. Percentages of control free heme are shown based on means from two independent assays.</p

Hydrogen bonds are crucial between the BC loop of falstatin and cysteine protease.

<p>Hemoglobin and fluorescence peptide hydrolysis were done in the presence of wild type peptide (LDSV<b>N²⁸⁷</b>G<b>N²⁸⁹</b>G<b>F²⁹¹</b>VW), mutant peptides (LDSV<b>N²⁸⁷</b>G<b>A²⁸⁹</b>G<b>A²⁹¹</b>VW and LDSV<b>N²⁸⁷</b>G<b>A²⁸⁹</b>G<b>F²⁹¹</b>VW), and mutants of falstatin (Asn ²⁸⁷, Phe ³⁹⁷). The Inhibitory property of wild type peptide and mutant peptides (16 μM) based on a BC loop were assessed by using fluorescence peptide (Z-Leu-Arg-AMC) with VP2 (Fig. 4A) or FP3 (Fig. 4B).The percentage of fluorescence units were measured and calculated for the hydrolysis of the fluorogenic substrate (A, B). Similarly, the Inhibitory property of wild type peptide and mutant peptide were assessed by using spectrophotometer at 410 nm (Fig. 4C). Error bars are shown based on means from two independent assays. Hemoglobin hydrolysis by VP2 (Fig. 4D) and FP3 (Fig. 4E) were also assessed in the presence of wild type (LDSVN²⁸⁷GN²⁸⁹GF²⁹¹VW) and mutant peptide (LDSVN²⁸⁷GA²⁸⁹GA²⁹¹VW) under optimal conditions and hydrolysis pattern was observed in 15% SDS-PAGE.</p

Models depicting the interactions of wild type peptide and VP2 (A), FP3 (B), and FP2 (C).

<p>3D models depict interactions of the BC loop peptides (turquoise) with the active site (green) regions of VP2 (A), FP3 (B) and FP2 via hydrogen bonds and hydrophobic interactions.</p