title: Studies of the pattern recognition molecule H-ficolin: specificity and purification.
abstract: Ficolins are pattern recognition molecules of the innate immune system. H-ficolin is found in plasma associated with mannan-binding lectin-associated serine proteases (MASPs). When H-ficolin binds to microorganisms the MASPs are activated, which in turn activate the complement system. H-ficolin is the most abundant ficolin in humans, yet its ligand binding characteristics and biological role remain obscure. We examined the binding of H-ficolin to Aerococcus viridans as well as to a more defined artificial target, i.e. acetylated bovine serum albumin. A strict dependence for calcium ions and inhibition at high NaCl concentration was found. The binding to acetylated bovine serum albumin was inhibited by acetylsalicylic acid and sodium acetate as well as by N-acetylated glucosamine and galactosamine (GlcNAc and GalNAc) and glycine (GlyNAc). The binding to A. viridans was sensitive to the same compounds, but, importantly, higher concentrations were needed for inhibition. N-Acetylated cysteine was also inhibitory, but this inhibition was parallel with reduction in the oligomerization of H-ficolin and thus represents structural changes of the molecule. Based on our findings, we developed a procedure for the purification of H-ficolin from serum, involving PEG precipitation, affinity chromatography on Sepharose derivatized with acetylated serum albumin, ion exchange chromatography, and gel permeation chromatography. The purified H-ficolin was observed to elute at 700 kDa, similar to what we find for H-ficolin in whole serum. MASP-2 was co-purified with H-ficolin, and the purified H-ficolin·MASP-2 complex could activate complement as measured by cleavage of complement factor C4. This study extends our knowledge of the specificity of this pattern recognition molecule, and the purified product will enable further studies.
authors.affiliation: Department of Medical Microbiology and Immunology, Aarhus University, 8000 Aarhus, Denmark.
authors.forename: Rikke M
authors.initials: RM
authors.lastname: Zacho
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Jensen
authors.forename: Randi
authors.initials: R
authors.lastname: Terp
authors.forename: Jens C
authors.initials: JC
authors.lastname: Jensenius
authors.forename: Steffen
authors.initials: S
authors.lastname: Thiel
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry; D000116:Acetylgalactosamine / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D000117:Acetylglucosamine / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D056570:Aerococcus; D000818:Animals; D001241:Aspirin / Q000737:chemistry; D002417:Cattle; D003181:Complement C4 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D037582:Complement Pathway, Mannose-Binding Lectin / Q000502:physiology; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology; D037102:Lectins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology; D019346:Sodium Acetate / Q000737:chemistry
chemical_list: D003181:Complement C4; C109662:FCN3 protein, human; D006023:Glycoproteins; D037102:Lectins; D046912:Multiprotein Complexes; D012710:Serum Albumin, Bovine; D019346:Sodium Acetate; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases; D000116:Acetylgalactosamine; D001241:Aspirin; D000117:Acetylglucosamine; D000111:Acetylcysteine