The binding of EBA-140 Region II to GPC in Western blotting.

<p>Normal (N), Gerbich (Ge) RBCs membranes and purified RBCs glycophorins (GP) were fractionated in SDS-PAGE and transferred to NC. RBCs glycophorins were desialylated (GP’) by the incubation of the blots in sulfuric acid. The blots were overlaid with the solution of recombinant Region II detected with MoAb anti-myc (clone 9E10). The GPC, GPD and variant GPC Gerbich was detected with MoAbs specific to N-terminus (clone 1G4) or C-terminus (clone1F6) of GPC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref037" target="_blank">37</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref038" target="_blank">38</a>]. GPC is visible on the blots as a double band stained with MoAb 1G4 or a single band stained with MoAb 1F6 due to a partial degradation of GPC at the C-terminus; M, protein molecular weight standards.</p

Analytical size exclusion chromatography of the EBA-140 Region II on Superdex 200 column.

<p>Purified protein was analyzed by SDS-PAGE in the gel stained with CBB and Western blotting with anti-myc MoAb (clone 9E10); peak 1 corresponds to the Region II (13,5ml elution volume); MW, protein molecular weight standards; RII, Region II (~ 75 kDa); elution volumes of standards used in column calibration are marked by the dashed lines.</p

Flow cytometry analysis of EBA-140 Region II binding to normal and variant human RBCs Gerbich phenotype containing a deletion variant of GPC (Δ36–63 a.a.).

<p>The black line corresponds to the control (RBCs incubated with the heat denatured Region II and then with anti-Region II rabbit serum); the grey peak corresponds to the binding of Region II to RBCs; the grey line corresponds to RBCs binding of MoAb anti-Ge3 recognizing a.a. residues 43–51 of GPC (clone 3C4) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref038" target="_blank">38</a>], indicating normal or Gerbich-negative RBCs phenotype.</p

Circular dichroism spectrum (195–270 nm) of EBA-140 Region II.

<p>Solution of Region II (0.5 µM) in 50 mM Tris-HCl, 300 mM NaCl, 10% glycerin, pH 7.5 was used.</p

Expression and purification of the recombinant EBA-140 Region II.

<p>A)Western blotting of baculovirus-infected Sf9 cells at 50 h post-infection with MoAb anti-myc (clone 9E10); MW, protein molecular weight standards; L, cell lysate; S, soluble cell fraction; IS, insoluble cell fraction; CM, culture medium. B) Affinity purification of the recombinant EBA-140 Region II on Ni-NTA resin. SDS–PAGE of proteins in the gel stained by CBB; MW, protein molecular weight standards; L, cell lysate; CM, culture medium; FT, flow- through of resin. C) Western blotting of fractions eluted with 50mM (1,2) and 100mM (3, 4) imidazole, with MoAb anti-myc (clone 9E10); RII, Region II (~ 75 kDa).</p

Surface plasmon resonance analysis of human RBCs binding to the recombinant EBA-140 Region II.

<p>The solid lines correspond to RBCs binding to NTA flow cell covered with the functional Region II; the dashed lines correspond to RBCs binding to NTA flow cell covered with the denatured RII; analysis was performed in PBS buffer with 0.5% BSA, pH 7.2 on Biacore T200.</p

Structure of N-terminal fragments of the polypeptide chain of GPC and its deletion variants Yus and Gerbich type.

<p>Asterisks denote O- glycosylation sites, the black oval denote the N-glycan. The numbers of a.a. residues relate to their position in normal GPC. The location of regions coded by exons: 1, 2, 3, 4 is indicated.</p

Flow cytometry analysis of EBA-140 Region II binding to normal and variant recombinant forms of GPC (Yus and Gerbich) expressed in CHO cells.

<p>The black line corresponds to the control (CHO cells incubated with anti-Region II rabbit serum); the grey peaks correspond to the binding of Region II to transfected or untransfected CHO cells; the grey line corresponds to the binding of MoAb RB8, recognizing a.a. residues 13–17 of GPC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref037" target="_blank">37</a>], to recombinant forms of GPC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115437#pone.0115437.ref033" target="_blank">33</a>].</p

Flow cytometry analysis of EBA-140 Region II binding to the native and neuraminidase (RBCs/neu) or trypsin (RBCs/trypsin)-treated human erythrocytes (RBCs).

<p>The black line corresponds to control (RBCs incubated with the heat denatured Region II and then with anti-Region II rabbit serum); the grey line (peak) corresponds to Region II of EBA-140 antigen.</p

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

<div><p>Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of <i>Hafnia alvei</i> PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement <i>in vitro</i>. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions <i>in vitro</i>, it may have numerous consequences <i>in vivo</i>. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.</p></div