title: 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.
abstract: 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.
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Man-Kupisinska
authors.affiliation: Laboratory of Immunobiology of Infections, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.|Institute of Microbiology, Immunology and Biotechnology, University of Lodz, Lodz, Poland.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Michalski
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Maciejewska
authors.affiliation: Laboratory of Immunobiology of Infections, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Swierzko
authors.affiliation: Laboratory of Immunobiology of Infections, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Cedzynski
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Czeslaw
authors.initials: C
authors.lastname: Lugowski
authors.affiliation: Department of Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Lukasiewicz
mesh_terms: D005591:Chemical Fractionation / Q000379:methods*; D037222:Collectins / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007136:Immunoglobulins / Q000378:metabolism*; D037102:Lectins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism*; D008024:Ligands; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D000097604:Ficolins
chemical_list: D037222:Collectins; D007136:Immunoglobulins; D037102:Lectins; D008024:Ligands; D050606:Mannose-Binding Protein-Associated Serine Proteases; D008562:Membrane Glycoproteins; D011951:Receptors, Complement; D013917:Thrombin; D000097604:Ficolins; C518521:Colec11 protein, human; C032402:complement 1q receptor