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

• Published in: PloS one 2016-05, Vol.11 (5), p.e0156691-e0156691
• Main Authors: Man-Kupisinska, Aleksandra, Michalski, Mateusz, Maciejewska, Anna, Swierzko, Anna S, Cedzynski, Maciej, Lugowski, Czeslaw, Lukasiewicz, Jolanta
• Format: Article
• Language: English
• Subjects: Affinity; Affinity chromatography; Analysis; Binding; Biological activity; Biology; Biology and Life Sciences; Blood plasma; Blood serum; Cancer; Chemical Fractionation - methods; Chromatography; Collectins - metabolism; Crosstalk; E coli; Ficolins; Fucose; Hafnia alvei; Hafnium oxide; Human serum albumin; Humans; Hydroxyapatite; Immunity, Innate; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Immunoglobulins - metabolism; Immunology; Incubation; Infections; Influenza; L-fucose; Laboratories; Lectins; Lectins - blood; Lectins - isolation & purification; Lectins - metabolism; Ligands; Lipopolysaccharides; Mannose; Mannose-binding lectin; Mannose-Binding Protein-Associated Serine Proteases - metabolism; Mannose-binding protein-associated serine proteinase; Medicine and Health Sciences; Membrane Glycoproteins - metabolism; Microorganisms; Monoclonal antibodies; Pattern recognition; Plasma; Polyethylene glycol; Proteins; Receptors, Complement - metabolism; Recombinant proteins; Research and Analysis Methods; Serine; Serum albumin; Size exclusion chromatography; Thrombin - metabolism; Yeast
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0156691

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 Hafnia alvei 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 in vitro. 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 in vitro, it may have numerous consequences in vivo. 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.