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

IgA, IgG, IgM, and MBL tracking in SEC-HPLC fractions.

<p>Dot-blot analyses were performed using (A) anti-ficolin-3 mAb; (B) anti-IgA α chain, IgG γ chain, IgM μ chain polyclonal Ab; and (C) anti-MBL mAb. Fractions 22–25 were eluted from the TSKgel G3000SW column (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156691#pone.0156691.g001" target="_blank">Fig 1</a>) and concentrated 9-fold. IgA (D), IgG (E), and IgM (F) tracking in ficolin-3 complexes with dot-blotting. Samples: 1. Ficolin-3 preparation, concentrated eluate from G3000SW column (fractions 22–25); 2. 10-fold diluted ficolin-3 preparation; 3. 100-fold diluted ficolin-3 preparation; 4. Human plasma; 5. Recombinant ficolin-3.</p

Detection of ficolin-3, MASPs and Ig in plasma-derived ficolin-3 complexes.

<p>Analysis of ficolin-3 preparation (1 μg of final product) fractionated on a 10% polyacrylamide gel under reducing (lanes 1, 2, 5–8) or non-reducing (lanes 3–4) conditions. Coomassie Blue stain (lanes 1, 3) and Western blotting using anti-ficolin-3 (lanes 2, 4), anti-MASP-1 (lane 5), anti-MASP-2 (lane 6), and anti-MASP-3 (lane 7) antibodies. The last lane (8) is the strip 4 re-probed with anti-human IgG, IgA and IgM. MASP bands are marked with asterisks (*). A, b, c–heavy chains of IgM (a), IgA (b), IgG (c); d- ficolin-3 monomer; e- light chains of Ig. The positions of the molecular mass markers are indicated in kDa.</p

Concentration-dependent activity of plasma-derived ficolin-3/MASP-1 and ficolin-3/MASP-2 complexes to activate LP.

<p>(A) MASP-1 activity is expressed as the fluorescence of a product emerging due to the capacity of MASP-1 to cleave VPR-AMC substrate. (B) MASP-2 activity was determined by detection of C4 activation products deposition assay by specific antibodies. Conditions excluding activation of complement classical pathway were applied (Petersen et al., 2001).</p

Cleavage of complement components C3 and C4 by plasma-derived ficolin-3/MASP complexes.

<p>C3 (A) or C4 (B) was incubated with different concentrations of ficolin-3 preparation: 5 μg (lane 2), 2.5 μg (lane 3), 1.25 μg (lane 4), and control sample without ficolin-3 (lane 1). The cleavage of C3 and C4 was examined by SDS–PAGE and Coomassie Blue staining. M, molecular mass markers. Description of cleavage products is underlined.</p

SPR-based competition assay of recombinant ficolin-3 interaction with O-PS 1200 and IgG.

<p>Two-step SPR analysis of the interaction of immobilized recombinant ficolin-3 with 670 μM O-PS 1200 and 6.67 μM human IgG. No regeneration step was included between O-PS and IgG. Sensor chip: CM5. RU, resonance units.</p

SEC-HPLC chromatography of plasma-derived ficolin-3 complexes on TSKgel G3000SW.

<p>Presence of ficolin-1, ficolin-2, ficolin-3, MBL and IgA, IgG, IgM (denoted “Ig”) were detected by dot-blot analysis, where a positive result is indicated by “+”. The apparent molecular mass of thyroglobulin (670 kDa), ferritin (440 kDa), BSA (66 kDa), chymotrypsinogen A (25 kDa) and cytochrome C (12 kDa) was determined in a separate chromatographic run; V₀, void volume. Even fraction numbers are indicated below the chromatograph line. Absorbance was monitored at 280 nm.</p

SPR analysis of IgG binding to recombinant ficolin-3.

<p>Ficolin-3 was immobilized on a CM5 chip. Ficolin-3 concentrations are indicated adjacent to sensorgrams. RU, resonance units.</p

Ficolin-3 Complexes Recovery and MBL and Total Protein Concentration During the Purification Process.

<p>Ficolin-3 Complexes Recovery and MBL and Total Protein Concentration During the Purification Process.</p

Table_1_Interaction of Mannose-Binding Lectin With Lipopolysaccharide Outer Core Region and Its Biological Consequences.pdf

<p>Lipopolysaccharide (LPS, endotoxin), the main surface antigen and virulence factor of Gram-negative bacteria, is composed of lipid A, core oligosaccharide, and O-specific polysaccharide (O-PS) regions. Each LPS region is capable of complement activation. We have demonstrated that LPS of Hafnia alvei, an opportunistic human pathogen, reacts strongly with human and murine mannose-binding lectins (MBLs). Moreover, MBL–LPS interactions were detected for the majority of other Gram-negative species investigated. H. alvei was used as a model pathogen to investigate the biological consequences of these interactions. The core oligosaccharide region of H. alvei LPS was identified as the main target for human and murine MBL, especially l-glycero-d-manno-heptose (Hep) and N-acetyl-d-glucosamine (GlcNAc) residues within the outer core region. MBL-binding motifs of LPS are accessible to MBL on the surface of bacterial cells and LPS aggregates. Generally, the accessibility of outer core structures for interaction with MBL is highest during the lag phase of bacterial growth. The LPS core oligosaccharide–MBL interactions led to complement activation and also induced an anaphylactoid shock in mice. Unlike Klebsiella pneumoniae O3 LPS, robust lectin pathway activation of H. alvei LPS in vivo was mainly the result of outer core recognition by MBL; involvement of the O-PS is not necessary for anaphylactoid shock induction. Our results contribute to a better understanding of MBL–LPS interaction and may support development of therapeutic strategies against sepsis based on complement inhibition.</p